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Abstract:

Disclosed herein are methods and materials for prognosing survival of
lung cancer patients, the methods comprising the detection of gains and
losses of minimal common regions and/or genes associated with prognosis
and benefit of chemotherapy.

Claims:

1. A method for determining a lung cancer prognosis predicting tumour
responsiveness and/or likelihood of improved survival with chemotherapy
in a subject, the method comprising: (a) determining a genomic profile
comprising detecting one or more genomic alterations in one or more of
chromosomes 2, 11, 4, 5, 7, 9, 12, 17, 19, 20, 8, 1, 13, 16, 6 and/or 14
listed in Tables 1 to 11, in a biological sample from the subject;
wherein the prognosis is determined to be poor when the genomic profile
comprises a gain of all or part of one or more minimal common regions
(MCRs) and/or genes within chromosomes 1, 2, 11, 4, 5, 6, 7, 9, 12, 14,
16, 17, 19 and 20, listed as associated with poor prognosis in Tables 1,
2, 5, 9, 10, and 11, and/or a loss of all or part of one or more MCRs
and/or genes within chromosomes 1, 5, 8, 13 and 16 listed as associated
with poor prognosis in Tables 3 and 7; and the prognosis is determined to
be good when the genomic profile comprises a genomic gain of all or part
of a MCR and/or gene within chromosome 8 listed as associated with good
prognosis in Table 6; and/or a loss of one or more MCRs and/or genes
within chromosome 6 or 14 listed as associated with good prognosis in
Table 8, relative to a control.

2. (canceled)

3. The method of claim 2, wherein the gain comprises a gain in all or
part of one or more of Table 11 genes FGF3, FAM112B, TSFM, NUP107 and/or
MDM2; or wherein the MCR listed as associated with poor prognosis is
selected from a MCR listed in Table 10.

4. The method of claim 1 comprising after step (a) the step: (b)
comparing the genomic profile with one or more controls.

5. (canceled)

6. The method of claim 1, wherein the prognosis is determined to be poor
when the genomic profile comprises a gain of all or part of a gene listed
in Table 5, 9, and/or 11 associated with poor prognosis and/or comprises
a loss of all or part of a gene listed in Table 7, and the prognosis is
determined to be good when the genomic profile comprises a gain of all or
part of gene listed in Table 6 or a loss of all or part of a gene listed
in Table 8 relative to the control.

7. The method of claim 1, wherein the method of determining a genomic
profile comprises: determining a hybridization pattern using one or more
chromosomal probes in the biological sample from the subject, wherein the
one or more probes hybridze specifically to one or more MCRs and/or genes
listed in Tables 1 to 11.

8. (canceled)

9. The method of claim 6, wherein the gain associated with good prognosis
comprises all or part of RAB11FIP1 and/or the loss associated with good
prognosis comprises all or part of a gene listed in Table 8.

10. The method of claim 4, wherein the one or more controls comprise a
control copy number such as centromere copy number or a control gene on
the same or different chromosome.

15. The method of claim 1, comprising detecting the expression level of a
gene listed in Table 5, 6, 7, 8, 9 and/or 11, wherein the expression
level of the gene all or partly gained or lost is increased or decreased
respectively, relative to a control expression level.

16. (canceled)

17. The method of claim 1 for selecting a treatment regimen for a subject
with lung cancer, the method comprising: (a) determining a genomic
profile comprising detecting a genomic alteration in one or more genes
selected from Table 5, 9 and/or 11 and/or 7 in a biological sample from
the subject; (b) selecting a treatment for the subject by comparing the
genomic profile with one or more controls, wherein the treatment selected
comprises chemotherapy when the genomic profile comprises a gain of all
or part of one or more genes associated with improved survival with
chemotherapy including the following genes: MFSD7, D4S234E, ACOX3,
SRD5A1, AQP2, ACCN2, SLC11A2, SCN8A, KRT81, KRT1, ESPL1, NPFF, ATP5G2,
HOXC11, NEUROD4, ZBTB39, KIAA0286, INHBE, MARS, B4GALNT1, TSFM, DNMT3B,
BAALC, ANGPT1, MYC, WISP1, KRT81, KRT1, NEUROD4, PA2G4, GUCA2A, PPIH,
LEPRE1, CR623026, C1orf50, DQ515898, DQ515897, MYC FGF3, KRT81, KRT1,
FAM112B, B4GALNT1, CENTG1, and/or BCL11B; and/or a loss of all or part of
one or more genes associated with improved survival with chemotherapy
including the following genes: RHOC, ATP2C2, ZDHHC7, COC4I1, FOXF1
relative to the control and/or wherein the treatment comprises a
non-chemotherapy treatment and/or a non-platinum analog, a vinca alkyloid
or a combination thereof chemotherapy treatment, when the genomic profile
comprises a gain of all or part of one or more of AK024870 and CPSF6.

18. The method of claim 1, wherein the biological sample is selected from
the group consisting of lung tissue, lung cells, lung biopsy and sputum,
including formalin fixed, paraffin embedded and fresh frozen specimens.

27. The method of claim 1 for determining a likelihood of improved
survival in a lung cancer subject who was or is receiving a
chemotherapeutic treatment, comprising determining the presence or
absence of a gain or loss of all or part of a MCR and/or gene associated
with improvement with chemotherapy, predicting the likelihood of improved
survival and/or predisposition to platinum analogs, vinca alkaloids
and/or a combination thereof according to the presence or absence of the
MCR or gene gain or loss compared to a control, wherein detecting a MCR
and/or gene associated with improvement with chemotherapy predicts
likelihood of improved survival compared to a control having the same
gain or loss who has not received and/or is not receiving chemotherapy,
and/or is indicative of a favourable predisposition of the subject to
respond to platinum analogs, vinca alkaloids and/or a combination
thereof.

28. (canceled)

29. The method of claim 1, for treating a subject with lung cancer
comprising determining the presence or absence of a gain or loss of a MCR
or gene associated with improvement with chemotherapy in a subject with
lung cancer and administering chemotherapy to a subject with at least one
gain or loss associated with improvement with chemotherapy.

30. The method of claim 29 wherein the chemotherapy is a platinum analog,
a vinca alkaloid or a combination thereof.

31. The method of claim 30 wherein the platinum analog is selected from
cisplatin, paraplatin, carboplatin, oxaliplatin and satraplatin in either
IV or oral form and/or wherein the vinca alkyloid is selected from
vinorelbine, vincristine, vinblastine, vindesine and vinflunine in either
IV or oral form.

32. (canceled)

33. A composition comprising two or more detection agents for detecting
the presence or absence of a MCR and/or gene gain or loss associated with
prognosis, wherein each detection agent comprises a hybridization probe;
or a primer and/or a primer pair for amplifying one or more genomic
alterations listed in Tables 1 to 11 for use in the method of claim 1.

39. A kit for determining lung cancer prognosis and/or tumour
responsiveness according to claim 1 in a subject, the kit comprising two
or more detection agents probe, wherein the two or more detection agents
are each a probe to a MCR and/or gene listed in Tables 1 to 11.

40. The kit of claim 39, wherein each detection agent comprises one or
more gene expression probes, or a set of probes specific for a gene
expression product of a gene listed in Tables 5, 6, 7, 8, 9 and/or 11, or
an array with one or more probes for one or more MCRs or genes gained or
lost described herein and labeling reagents for labeling the subject
sample DNA comprises a primer set for amplifying all or part of a MCR or
gene listed in any one of Tables 1 to 11 associated with prognosis,
optionally comprising one or more of the primers listed in Table 12.

41. (canceled)

42. (canceled)

43. (canceled)

44. (canceled)

45. (canceled)

46. The method of claim 1 wherein the method comprises (a) determining a
hybridization pattern of a chromosomal probe or a set of chromosomal
probes in a biological sample from the subject, wherein the probe or
probeset is targeted to all or part of one or more MCRs listed in the
provided tables, including but not limited to NRG4 on the short arm of
chromosome 1 (1p), NRG58 on 8q, NRG74 on 11q, NRG79 on 12q, NRG80 on 12q,
NRG81 on 12q, NRG82 on 12q, and/or NRG89 on 14q; (b) determining the
prognosis and/or predicting the response to chemotherapy for a patient
with lung cancer based on the hybridization pattern, wherein the
prognosis is determined to be poor without chemotherapy when the
hybridization pattern indicates a gain of DNA copy number at an MCR on
11q and/or a gain at an MCR on 12q and/or a gain at an MCR on 14q
relative to a control; and/or the prognosis is determined to be good when
treated with chemotherapy when the hybridization pattern indicates a gain
of DNA copy number within an MCR on 1p and/or 8q and/or 11q and/or 12q
and/or 14q.

Description:

[0001] This application claims the benefit of 35 U.S.C. 119 based on the
priority of co-pending U.S. provisional patent applications 61/171,356,
filed Apr. 21, 2009 and 61/171,687 filed Apr. 22, 2009, each of which are
herein incorporated by reference in their entirety.

[0003] Lung cancer is the leading cause of cancer death in Canada
(Canadian Cancer Society, 2008). Even after complete surgical resection
of stage I-III non-small-cell lung cancer (NSCLC), approximately half of
patients will recur and die within 5 years (Azzoli et al, 2008). Current
NSCLC clinicopathologic staging is not adequate to accurately predict
which patients will be cured by surgery alone, and which patients with
high risk of disease recurrence and mortality need adjuvant therapies.

[0004] Many studies have examined gene and protein expression patterns in
NSCLC for refining the prognostication and treatment of the disease, with
some success. However, the impact on patient survival and response to
therapy for gene copy number alterations (amplifications and deletions)
is an area that has not been well studied in this regard.

[0005] Gene copy number changes are worthy of close examination in NSCLC,
because they have been shown to provide important information in other
malignancies. HER2/neu amplification in breast cancer is the best-known
example, where it has been shown to impart a much worse survival (Slamon
D J et al, 1987) as well as predict the response to systemic
chemotherapies (Dhesy-Thind et al, 2007). B-cell chronic lymphocytic
leukemia/lymphoma is another well-studied example; deletions at 13q14
have been shown to be associated with prolonged survival, whereas
deletions at 11q22-23 and at the TP53 locus on chromosome 17p have both
been associated with a poor prognosis (Jaffe, 2003). Many similar
discoveries of associations of gene copy number gains and losses with
patient outcome are rapidly being discovered in many different
malignancies. Detailed mechanistic studies may help further our
understanding of the pathobiology and ultimately provide better
treatments for patients.

[0006] Microarray comparative genomic hybridization (array-CGH) is a
relatively new technique, capable of detecting gains and losses of
genomic material at high-resolution across the genome, that has begun to
revolutionize this body of knowledge. Recent studies have demonstrated
the ability of array-CGH to subtype breast carcinomas (Climent et al,
2007a), DLBCL (Tagawa et al, 2005), CLL (Patel et al, 2008), and gliomas
(Idbaih et al, 2008) into distinct groups based on their pattern of gains
and losses. Many studies have shown an impact of specific gains or losses
on patient survival, including colorectal carcinoma (Kim et al, 2006),
gastric adenocarcinoma (Weiss et al, 2004), breast carcinoma (Han et al,
2006), mantle cell lymphoma (Rubio-Moscardo et al, 2005), diffuse large B
cell lymphoma (Chen et al., 2006; Tagawa et al, 2005), neuroblastoma
(Tomioka et al, 2008), and gliomas (Idbaih et al, 2008). In one study of
breast carcinomas from patients enrolled in a clinical trial, the loss of
a specific region of chromosome 11q was shown to be associated with a
good response to anthracycline-based chemotherapy (Climent et al, 2007b).
Two previous studies have shown that reliable array-CGH profiles can be
obtained using archival formalin-fixed, paraffin-embedded (FFPE) tissues
(Fenesterer et al, 2007; Mayr et al, 2006). This is very important, as it
allows this powerful technique to be performed on the vast quantity of
routinely handled and archived surgical specimens of diagnostic
laboratories.

[0007] Similar to other epithelial malignancies, the karyotypes of NSCLC
show multiple and complex chromosomal aberrations, resulting in net gain
or loss of genetic material, indicative of genomic instability (Balsara
et al, 2002). The imbalance profiles of the histologic subtypes of NSCLC
(adenocarcinoma, squamous carcinoma, and large cell carcinoma) are
similar, with frequent gains involving 5p, 8q, 3q, and 1q, frequent
losses at 3p, 8p, 9p, 13q, and 17p, and often showing polyploidy (in the
range of 58-102 chromosomes per cell) (Hoglund et al, 2004).
Amplifications are commonly observed in the form of double minutes.
Knowledge of the order or progression of these aberrations is scarce, but
some have speculated that early events include trisomy 7, loss at 3p, and
trisomy 12. Gains at both 7q and 8q have been associated with higher
stage tumours with positive nodal involvement and higher tumour grade,
and 20q13 gains have been linked with invasiveness in adenocarcinoma
(AC).

[0008] Genes reported to be amplified have included MYC, TERT, cyclin D1,
and EGFR. Increased epidermal growth factor (EGFR) copy number are seen
in 8-30% of patients by FISH and qPCR, and are often seen in conjunction
with mutations in the EGFR tyrosine kinase domain (Thomas et al, 2006).
Both amplification and mutations are associated with a specific
demographic: East Asian, female, never smokers, with adenocarcinomas
often showing a bronchioloalveolar histologic pattern (Sequist et al,
2007). Studies have shown that these patients tend to have a rapid,
dramatic, and durable response to gefitinib, a drug specifically designed
to inhibit the tyrosine kinase signaling activity of EGFR (Cappuzo et al,
2005; Takano et al, 2005; Hirsch et al, 2007). This finding is an
exciting example of how the identification of genetic events such as
amplification and mutation can lead to effective targeted therapies.
Strategies such as this could eventually lead to effective individualized
chemotherapy designed against many other altered pathways.

[0009] P63 amplification has also been shown to have a prognostic utility
in NSCLC. Massion et al (2003) applied FISH and immunohistochemistry to
detect P63 gene amplification and protein expression in tissue
microarrays containing 217 NSCLC samples. They found that P63 copy number
>=3 and increased immunostaining intensity were both significantly
associated with a better survival.

[0010] Array-CGH has allowed researchers to study gene copy number
aberrations in even greater detail (Dehan et al, 2007; Choi et al, 2007;
Zhao et al, 2005; Jiang et al, 2004). The high resolution of this
technique is clarifying genomic amplification and deletion to regions
often containing only a few genes, as well as identifying small,
previously undetected aberrations. As a result, the list of genes
implicated in NSCLC pathobiology is growing rapidly. Tonon et al. (2005)
identified 93 minimal common regions (MCRs) of aberration in NSCLC
tumours and cell lines, 21 of which spanned less than 0.5 Mb with a
median of 5 genes in each, with virtually all genes previously implicated
in NSCLC pathogenesis present within these regions, as well as many novel
candidate genes. Patterns of aberrations were similar between
adenocarcinoma (AC) and squamous carcinoma (SqC); supervised or
unsupervised clustering was unable to differentiate the two. Only the
amplification on 3q26-29 has been targeted significantly in SqC, similar
to previous findings by Massion et al (2002).

[0011] In a large study of 371 adenocarcinomas using SNP array-CGH, Weir
et al (2007) identified 26 recurrent large-scale events involving gain or
loss of at least half of a chromosome, together comprising more than half
of the human genome. In addition, 31 focal amplifications and homozygous
deletions were identified, including multiple novel candidate genes. One
of the homozygously deleted genes was PTPRD, a tyrosine phosphatase. Upon
sequencing of this gene, somatic mutations were found in 11 of 188
samples, indicating a role in PTPRD dysregulation in a subset of ACs. The
most common focal amplification, at 14q13.3, contained no known
proto-oncogene. Biological studies using RNAi knockout of the 2 genes
found within this region identified that NKX2-1 as a key factor in the
growth of cell lines with 14q amplifications.

[0012] Findings such as these highlight the power and utility of array-CGH
for finding specific molecular aberrations in subsets of NSCLC. However,
lacking in the literature are studies correlating these genomic events
with patient outcome. Shibata et al (2005) studied 55 ACs and were able
to split the tumours into 3 groups by unsupervised hierarchical
clustering. These clusters were associated with distinct genetic
alterations and showed an association with smoking history and gender,
but no association with stage or disease-free survival. However, two
specific alterations did show an association with disease-free survival
on multivariate analysis: loss on 13q14.1 and gain of 8q24.2 were both
associated with a poor outcome.

[0014] Disclosed herein are genes and genomic regions, the gain or loss of
which are associated with prognosis of lung cancer. A subset are
associated with significant improvement when administered chemotherapy.
Detecting the gains and losses are useful for determining a prognosis for
a subject with lung cancer and for guiding treatment selection.

[0015] Accordingly in an aspect, the disclosure provides a method for
determining a lung cancer prognosis in a subject, the method comprising:
(a) determining a genomic profile comprising detecting the presence or
absence of one or more genomic alterations in one or more of chromosomes
2, 11, 4, 5, 7, 9, 12, 17, 19, 20, 8, 1, 13, 16, 6 and/or 14 listed in
Tables 1-11 in a biological sample from the subject; wherein the
prognosis is determined to be poor when the genomic profile comprises a
gain of all or part of one or more minimal common regions (MCRs) and/or
genes within one or more of chromosomes 1, 2, 11, 4, 5, 6, 7, 9, 12, 14,
16, 17, 19 and/or 20, listed as associated with poor prognosis (e.g.
associated with survival) in Tables 1, 2, 5, 9, 10, and/or 11 and/or a
loss of all or part of one or more MCRs and/or genes within one or more
chromosomes 1, 5, 8, 13 and/or 16 listed as associated with poor
prognosis in Table 3 and/or 7 and the prognosis is determined to be good
when the genomic profile comprises a genomic gain of all or part of a MCR
and/or gne within chromosome 8 listed as associated with good prognosis
in Table 6 and/or a loss of all or part of one or more MCRs and/or genes
within chromosome 2, 6, 9 and/or 14 listed as associated with good
prognosis in Tables 8 relative to a control.

[0016] In an embodiment, the method comprises: (a) determining a genomic
profile comprising detecting the presence or absence all or part of one
or more genomic alterations in one or more of chromosomes 2, 11, 4, 5, 7,
9, 12, 17, 19, 20, 8, 1, 13, 16, 6 and/or 14 and/or genes listed in
Tables 1-11 in a biological sample from the subject; (b) determining the
lung cancer prognosis for the subject by comparing the genomic profile
with one or more controls, wherein the prognosis is determined to be poor
when the genomic profile comprises a gain of all or part of one or more
minimal common regions (MCRs) and/or genes within chromosomes 1, 2, 11,
4, 5, 6, 7, 9, 12, 14, 16, 17, 19 and/or 20, listed as associated with
poor prognosis in Tables 1, 2, 5, 9, 10, and/or 11 and/or a loss of all
or part of one or more MCRs within chromosomes 1, 5, 8, 13 and/or 16
listed as associated with poor prognosis in Tables 3 and/or 7; and the
prognosis is determined to be good when the genomic profile comprises a
genomic gain of all or part of a MCR and/or gene within chromosome 8
listed as associated with good prognosis in Table 6 and/or a loss of all
or part of one or more MCRs and/or genes within chromosome 2, 6, 9 and/or
14 listed as associated with good prognosis in Table 6 and/or 8 relative
to the control.

[0017] In an embodiment, the method comprises obtaining a biological
sample for determining the genomic profile.

[0018] In an embodiment, the prognosis is determined to be poor when the
genomic profile comprises a gain of all or part of a gene listed in Table
5, and/or comprises a loss of all or part of a gene listed in Table 7,
and the prognosis is determined to be good when the genomic profile
comprises a gain of all or part of a gene listed in Table 6 and/or a loss
of all or part of a gene listed in Table 8 relative to the control. In an
embodiment, the prognosis is determined to be poor when the genomic
profile comprises a gain of all or part of a gene listed in Table 9,
and/or 11 identified as associated significantly and/or trending to
significance with poor prognosis. In an embodiment, the gene associated
with prognosis is a gene that shows a trend to significance. In another
embodiment, the gene associated with prognosis is a gene with a
significant association.

[0019] In an embodiment, the presence or absence of a genomic alteration
is determined using a chromosomal probe and detecting a hybridization
pattern.

[0020] In another embodiment, the prognosis is determined to be poor when
the hybridization pattern indicates a gain of all or part of a MCR or a
gene listed in Table 1, 2, 5 and/or 9-11 (for genes identified as
associated with poor prognosis) and/or loss of all or part a MCR or gene
listed in Table 3 and/or 7. In a further embodiment, the gain comprises
all or part of a gene listed in Table 5 and/or the loss comprises all or
part of a gene listed in Table 7. In yet another embodiment, the gain
comprises all or part of one or more of genes listed in Tables 9 and/or
11.

[0022] In another embodiment, the prognosis is determined to be good when
the hybridization pattern indicates a gain of all or part of a MCR within
chromosome 8 associated with good prognosis and/or a loss of all or part
of one or more MCRs within chromosome 6 or 14 associated with good
prognosis relative to a control. In an embodiment, the gain comprises all
or part of RAB11FIP1 and/or the loss comprises all or part of a gene
listed in Table 8.

[0023] In an embodiment, the presence or absence of a genomic alteration
is determined using a chromosomal probe. In another embodiment, the
control is a control copy number, centromere copy number or a control
gene on the same or different chromosome.

[0024] In another aspect, the disclosure includes a method for determining
a likelihood of improved survival or response with chemotherapy treatment
comprising detecting a gain of all or part of a MCR or gene listed in
Tables 1, 2, 5, 9, 10 and/or 11 associated with improved response to
chemotherapy, wherein a gain indicates the subject has a good prognosis
when treated with chemotherapy relative to a subject not treated with
chemotherapy.

[0025] In another aspect, the disclosure includes a method for determining
tumour responsiveness to a chemotherapy treatment comprising detecting a
gain of all or part of one or more of the genes listed in Tables 1, 2, 5,
9 or 11 associated with improved response to chemotherapy, wherein a gain
indicates the tumour is likely responsive to treatment with chemotherapy
relative to a tumour not comprising the gain.

[0026] In an embodiment, the gain associated with improved survival with
chemotherapy or improved tumor responsiveness is a gain of all or part of
one or more of the following genes: MFSD7, D4S234E, ACOX3, SRD5A1, AQP2,
ACCN2, SLC11A2, SCN8A, KRT81, KRT1, ESPL1, NPFF, ATP5G2, HOXC11, NEUROD4,
ZBTB39, KIAA0286, INHBE, MARS, B4GALNT1, TSFM, DNMT3B.

[0027] In another embodiment, the gain associated with improved survival
with chemotherapy or improved tumor responsiveness is a gain of all or
part of one or more of the following genes: BAALC, ANGPT1, MYC, WISP1,
KRT81, KRT1, NEUROD4, and/or PA2G4 (e.g. Table 9 genes associated with
improved response to chemotherapy). In a further embodiment, the gain
associated with improved survival with chemotherapy or improved tumor
responsiveness is a gain of all or part of one or more of the following
genes: GUCA2A, PPIH, LEPRE1, CR623026, C1orf50, DQ515898, DQ515897, MYC
FGF3, KRT81, KRT1, FAM112B, B4GALNT1, CENTG1, BCL11B (e.g. Table 11 genes
associated with improved response to chemotherapy).

[0028] In another aspect, the disclosure includes a method for determining
a likelihood of improved survival with chemotherapy treatment comprising
detecting a loss of all or part of a MCR and/or gene listed in Tables 3,
4, 7 and/or 8 associated with improved response to chemotherapy, wherein
the loss indicates the subject has a good prognosis when treated with
chemotherapy relative to a subject not treated with chemotherapy.

[0029] In another aspect, the disclosure includes a method for determining
tumour responsiveness to a chemotherapy treatment comprising detecting a
loss of all or part of a MCR and/or gene listed in Tables 3, 4, 7 and/or
8 associated with improved response to chemotherapy, wherein the loss
indicates the tumour is likely responsive to treatment with chemotherapy
relative to a tumour not comprising the loss.

[0030] In an embodiment, the loss is of all or part of one of the
following genes: RHOC, ATP2C2, ZDHHC7, COC4I1, and/or FOXF1.

[0032] In another aspect, the method further comprises detecting the
expression level of a gene listed in Table 5, 6, 7, 8, 9 and/or 11. For
example, the expression level of a gene associated with prognosis and/or
response to chemotherapy can be detected for predicting a prognosis
and/or for predicting tumour responsiveness. In an embodiment, the
expression level of the gene all or partly gained or lost is increased or
decreased respectively, relative to a control expression level wherein
increased expression of a gene gain listed in Table 5 and/or decreased
expression of a gene listed in Table 7 indicates poor prognosis without
chemotherapy, and/or increased expression of a gene listed in Table 6
and/or decreased expression of a gene listed in Table 8 indicates good
prognosis. In a further embodiment, the expression level of a gene listed
in Table 9 or 11 is detected.

[0033] Another aspect comprises a method for determining a lung cancer
prognosis in a subject, the method comprising: (a) determining a
hybridization pattern of a chromosomal probe or a set of chromosomal
probes in a biological sample from the subject, wherein the probe or
probeset is targeted to all or part of one or more MCRs listed in the
provided tables, including but not limited to NRG4 on the short arm of
chromosome 1 (1p), NRG58 on 8q, NRG74 on 11q, NRG79 on 12q, NRG80 on 12q,
NRG81 on 12q, NRG82 on 12q, and/or NRG89 on 14q; (b) determining the
prognosis and/or predicting the response to chemotherapy for a patient
with lung cancer based on the hybridization pattern, wherein the
prognosis is determined to be poor without chemotherapy when the
hybridization pattern indicates a gain of DNA copy number at an MCR on
11q and/or a gain at an MCR on 12q and/or a gain at an MCR on 14q
relative to a control; and/or the prognosis is determined to be good when
treated with chemotherapy when the hybridization pattern indicates a gain
of DNA copy number within an MCR on 1p and/or 8q and/or 11q and/or 12q
and/or 14q.

[0034] In an embodiment, the gain of DNA copy number is at or within an
MCR located at approximately base-pair positions 41265460 to about
43221579 on the short arm of chromosome 1, and is indicative of a good
prognosis with chemotherapy.

[0035] In another embodiment, the gain of DNA copy number is at an MCR
located at approximately base-pair positions 128289292 to about 128936748
on the long arm of chromosome 8 is indicative of a good prognosis with
chemotherapy.

[0036] In another embodiment, the gain of DNA copy number is at or within
an MCR located at approximately base-pair positions 68572940 to about
70388868 on the long arm of chromosome 11 is indicative of a good
prognosis with chemotherapy.

[0037] In another embodiment, the gain of DNA copy number is at or within
an MCR located at approximately base-pair positions 50731457 to about
51457372 on the long arm of chromosome 12 is indicative of a good
prognosis with chemotherapy.

[0038] In another embodiment, the gain of DNA copy number is at or within
an MCR located at approximately base-pair positions 52696908 to about
53538441 on the long arm of chromosome 12 is indicative of a good
prognosis with chemotherapy.

[0039] In another embodiment, the gain of DNA copy number is at or within
an MCR located at approximately base-pair positions 55933813 to about
57461765 on the long arm of chromosome 12 is indicative of a good
prognosis with chemotherapy.

[0040] In another embodiment, the gain of DNA copy number is at or within
an MCR located at approximately base-pair positions 96994959 to about
99058653 on the long arm of chromosome 14 is indicative of a good
prognosis with chemotherapy.

[0041] Another aspect relates to a method of selecting a treatment regimen
for a subject with lung cancer, the method comprising: (a) determining a
genomic profile comprising detecting a genomic alteration of all or part
of one or more MCRs and/or genes selected from MCRs and genes identified
herein associated with survival with chemotherapy, for example as listed
in Table 1, 2, 3, 5, 7, 9, 10 and/or 11; in a biological sample from the
subject; and (b) selecting chemotherapy when a gain or loss associated
with improved survival with chemotherapy is detected and/or not selecting
chemotherapy and/or selecting a non-chemotherapy and/or a non-platinum
analog-, a vinca alkyloid- and/or combination thereof chemotherapy, when
a gene associated with worse survival with chemotherapy.

[0042] In an embodiment, the method comprises: (a) determining a genomic
profile comprising detecting a genomic alteration in one or more genes
selected from Table 5 and/or 7 in a biological sample from the subject;
(b) selecting chemotherapy for the subject when the genomic profile
comprises a gain of all or part of one or more of the following genes:
MFSD7, D4S234E, ACOX3, SRD5A1, AQP2, ACCN2, SLC11A2, SCN8A, KRT81, KRT1,
ESPL1, NPFF, ATP5G2, HOXC11, NEUROD4, ZBTB39, KIAA0286, INHBE, MARS,
B4GALNT1, TSFM, and/or DNMT3B; and/or a loss of all or part of one or
more of the following genes: RHOC, ATP2C2, ZDHHC7, COC4I1, and/or FOXF1
relative to a control.

[0043] In another embodiment, the method comprises: (a) determining a
genomic profile comprising detecting a genomic alteration in one or more
genes selected from Table 9 and/or 11 in a biological sample from the
subject; (b) selecting chemotherapy for the subject when the genomic
profile comprises a gain of all or part of one or more of the following
genes: BAALC, ANGPT1, MYC, WISP1, KRT81, KRT1, NEUROD4, and/or PA2G4
(e.g. Table 9 genes associated with improved response to chemotherapy).
In a further embodiment, the gain associated with improved survival with
chemotherapy or improved tumor responsiveness is a gain of all or part of
one or more of the following genes: GUCA2A, PPIH, LEPRE1, CR623026,
C1orf50, DQ515898, DQ515897, MYC, FGF3, KRT81, KRT1, FAM112B, B4GALNT1,
CENTG1, and/or BCL11B (e.g. Table 11 genes associated with improved
response to chemotherapy). In an embodiment, the method comprises not
selecting chemotherapy and/or not selecting a chemotherapeutic regimen
comprising a platinum analog, a vinca alkyloid and/or a combination
thereof e.g. selecting a non-chemotherapy and/or a non-platinum analog-,
vinca alkyloid- or combination thereof chemotherapy, when a gain at
AK024870, CPSF6 is detected.

[0044] In certain embodiments, the biological sample is selected from the
group consisting of lung tissue, lung cells, lung biopsy and sputum,
including formalin fixed, paraffin embedded and fresh frozen specimens.

[0045] Also provided is a method for determining a lung cancer prognosis
in a subject, the method comprising: detecting the presence or absence of
a genomic alteration at a locus identified in Tables 1, 2, 3, 4, 5, 6, 7,
8, 9, 10 and/or 11 in a biological sample from the subject, wherein the
prognosis is determined to be poor in the absence of chemotherapy when a
gain of all or part of a MCR listed in Tables 1 and/or 2 or a gene listed
in Table 5 and/or a loss of all or part of a MCR listed in Table 3 and/or
gene listed in Table 7 is detected; and the prognosis is determined to be
good when a gain of all or part of a MCR or gene listed in Table 6 and/or
loss of all or part of a MCR or gene in Table 4 and/or 8 is detected
relative to a control. In another embodiment, a gain of all or part of a
MCR listed in Table 10 and/or a gene listed in Table 9 and/or 11, wherein
the prognosis is determined to be poor in the absence of chemotherapy
when a gain associated with poor prognosis (including trending to poor
prognosis) is detected.

[0046] In an embodiment, the presence or absence of a gain of DNA copy
number is detected at an MCR at 12q at or within basepair positions
50731457 to 51457372, and/or 12q at or within basepair positions 52696908
to 53538441, and/or 12q at or within basepair positions 55933813 to
57461765, and/or 12q at or within basepair positions 64438067 to
68503251, and/or 14q at or within basepair positions 96994959 to
99058653. In another embodiment, the presence or absence of a gain of DNA
copy number is detected at an MCR at 12q at or within basepair positions
50731457 to 51457372. In another embodiment, the presence or absence of a
gain of DNA copy number is detected at an MCR at 12q at or within
basepair positions 52696908 to 53538441. In another embodiment, the
presence or absence of a gain of DNA copy number is detected at an MCR at
12q at or within basepair positions 55933813 to 57461765. In another
embodiment, the presence or absence of a gain of DNA copy number is
detected at an MCR at 12q at or within basepair positions 64438067 to
68503251. In another embodiment, the presence or absence of a gain of DNA
copy number is detected at an MCR at 14q at or within basepair positions
96994959 to 99058653. In yet a further embodiment the genomic alteration
comprises all or part of a MCR listed in Table 1, 2, 3, 4 and/or 10.

[0047] In an embodiment, the presence or absence of a DNA copy number
alteration at for example, the position of a gene located within the MCRs
gained or lost, for example genes within the MCRs listed in any one of
Tables 1 to 11 are detected. In an embodiment, the presence or absence of
a DNA copy number alteration at the position of a gene from the group
consisting of KRT1, ESPL1, NPFF, ATP5G2, HOXC11, and/or genes within an
MCR located between 50-57 Mb on chromosome arm 12q (e.g. MCR IDs NRG79,
NRG80, NRG81, NRG82) is detected. In another embodiment, the presence or
absence of a gene from the group consisting of ITGA7, CDK2, BCDO2, ERBB3,
DLST, PA2G4, ZBTB39 and/or TSFM which are comprised in the MCRs at
55.2-55.6 Mbp on chromosome arm 12q are detected. In another embodiment,
the gene detected is all or part of a gene listed in Table 9 and/or 11.

[0048] Another aspect provides a method of predicting response to a
chemotherapeutic treatment in a subject with lung cancer comprising
detecting the presence or absence of a gain or loss of all or part of a
MCR or a gene in any one of Tables 1-11, predicting the response to the
chemotherapeutic according to the presence or absence of the MCR or gene
gain or loss compared to a control, wherein detecting a MCR or gene
associated with improvement with chemotherapy predicts chemotherapy will
be efficacious, for example will, improve survival and/or wherein
detecting a MCR and/or gene not associated with improvement with
chemotherapy predicts no response to chemotherapy.

[0049] A further aspect provides a method of determining a likelihood of
improved survival in a lung cancer subject who was or is receiving a
chemotherapeutic treatment, comprising determining the presence or
absence of a gain or loss of all or part of a MCR and/or gene associated
with improvement with chemotherapy, predicting the likelihood of improved
survival according to the presence or absence of the MCR and/or gene gain
or loss compared to a control, wherein detecting all or part of a gain
and/or loss of a MCR and/or gene associated with improvement with
chemotherapy predicts likelihood of improved survival compared to a
control having the same gain and/or loss who has not received and/or is
not receiving chemotherapy. In an embodiment, the presence of a gain
and/or loss associated with improvement with chemotherapy is indicative
of a favourable predisposition of the subject to respond to platinum
analogs, vinca alkyloids and/or a combination thereof.

[0051] Another aspect provides a method of treating lung cancer comprising
determining the presence or absence of a gain and/or loss of all or part
of a MCR and/or gene associated with improvement with chemotherapy in a
subject with lung cancer and administering chemotherapy to the subject
with at least one gain or loss associated with improvement with
chemotherapy.

[0052] In an embodiment, the chemotherapy is a platinum analog, a vinca
alkaloid or a combination thereof. In a further embodiment, the platinum
analog is selected from the group consisting of cisplatin, paraplatin,
carboplatin, oxaliplatin and satraplatin in either IV or oral form. In
another embodiment, the vinca alkaloid is selected from the group
vinorelbine, vincristine, vinblastine, vindesine and vinflunine in either
IV or oral form.

[0053] A further aspect relates to a composition comprising a detection
agent for detecting all or part of a MCR and/or gene gain or loss
associated with prognosis. In an embodiment, the composition comprises a
probe that binds and/or hybridizes with all or part of a MCR and/or a
gene described herein, and/or a primer or primer pair for amplifying a
polynucleotide comprising all or part of a MCR and/or gene associated
with prognosis described herein. In an embodiment, the probe is a BAC
clone listed in Table 13 and/or the primer is a primer listed in Table
12.

[0054] Yet a further aspect provided is a kit for determining lung cancer
prognosis in a subject. In and embodiment, the kit comprises a
chromosomal probe and/or a set of chromosomal probes, wherein the probe
or set comprises a probe to a MCR or part thereof listed in any one of
Tables 1 to 11 and/or a gene or part thereof listed in Tables 5, 6, 7, 8,
9 and/or 11. In another embodiment the kit comprises one or more gene
expression probes, wherein a probe is specific for a gene expression
product of a gene listed in Tables 5, 6, 7, 8, 9 and/or 11. In an
embodiment, the probes are labeled, optionally fluorescently labeled or
labelled with a chromagen. In another embodiment, the probes are
comprised in an array on a solid support. In yet a further embodiment,
the kit further comprising instructions that indicate prognosis is
determined to be poor when a hybridization pattern of the set of
chromosomal probes indicates a gain in all or part of a MCR in 12q,
and/or a gain in all or part of a MCR comprising all or part of a gene
listed in Table 5, 9 and/or 11 and/or a loss of all or part of a MCR
comprising all or part of a gene listed in Table 7, relative to control;
and/or to be good when a hybridization pattern of chromosomal probes
indicates a gain in all or part of a MCR comprising all or part of a gene
listed in Table 6 and/or a loss of all or part of a MCR comprising all or
part of a gene listed in Table 8; optionally wherein the control is
centromere copy number.

[0055] In an embodiment, the kit comprises a reagent for FISH analysis of
a MCR or a gene gain or loss described herein, for example, the kit
comprises a probe for a MCR or gene gain or loss described herein, for
example a BAC clone comprising all or part of a target MCR or gene,
including for example the BAC clones listed in Table 13 and/or labeling
reagents for labeling the probe. In a further embodiment, the kit
comprises a reagent for CGH analysis of a MCR or gene gain or loss
described herein, for example, the kit comprises an array with one or
more probes for detecting all or part of one or more MCRs or genes gained
or lost described herein and/or labeling reagents for labeling the
subject sample DNA. In a further embodiment, the kit comprises a reagent
for PCR such as quantitative or multiplex PCR, for example the kit
comprises a primer set for amplifying all or part of a MCR or gene
described herein associated with prognosis.

[0056] Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this disclosure pertains. Although methods and
materials similar or equivalent to those described herein can be used in
the practice or testing of methods and compositions described herein, a
few selected suitable methods and materials are described in more details
below. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety, including nucleic acid sequences identified by Entrez Gene ID,
unigene ID or other gene identifier number referred to herein and
particularly as provided in the Tables. In case of conflict, the present
specification, including definitions, will control. In addition, the
materials, methods, and examples are illustrative only and are not
intended to be limiting in any respect.

[0057] All embodiments of the disclosure, including those described under
different aspects of the disclosure, are contemplated to be combined with
other embodiments whenever applicable.

[0058] Other features and advantages of the present disclosure will become
apparent from the following detailed description and claims. It should be
understood, however, that the detailed description and the specific
examples while indicating preferred embodiments of the disclosure are
given by way of illustration only, since various changes and
modifications within the spirit and scope of the disclosure will become
apparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE DISCLOSURE

I. Definitions

[0059] The term "lung cancer" as used herein refers to cancers of the
tissues or cells of the lung including for example non-small cell lung
cancer (NSCLC), and small cell lung cancer (SCLC). The term could also be
used to refer to cancers that have arisen in the lung and have
metastasized to other sites (e.g. brain, liver, adrenals).

[0060] The term "non-small cell lung cancer" as used herein refers to
primary lung cancer that is distinguished from small cell lung cancer and
that is composed of multiple different types, including adenocarcinoma,
squamous cell carcinoma, large cell carcinoma and other less frequent
types.

[0061] The term "lung adenocarcinoma" and/or "lung ADC" and/or "pulmonary
ADC" as used herein refer to a type of lung cancer and comprises various
subtypes including bronchioloalveolar carcinoma (BAC) which is non
invasive and/or includes focal invasion and has good prognosis (2) and
invasive ADC including mixed type, which can have areas with BAC like
pattern and is referred to as invasive ADC with BAC features (AWBF).

[0062] The term "control" as used herein refers to a specific value or
dataset e.g., control expression level, control gene copy number,
reference expression profile or reference genomic profile according to
the context which a person skilled in the art would readily understand,
derived from one or more samples of a known subject class e.g., lung
cancer free class not having a MCR or a gene gain or loss described
herein, that is suitable for comparison to the value or dataset derived
from a subject sample. For example, the control can be a value or dataset
derived from tumor adjacent non-neoplastic normal tissue or tissue from a
disease free subject, e.g. for comparing to a lung cancer subject gene
expression profile. With respect to genomic alterations e.g. gains and
losses, the control can for example also refer to an internal control
e.g. the copy number of a non-altered region of the chromosome or a
different chromosome e.g. a chromosome with minimal variance in lung
cancer subjects, for example a chromosome not herein or previously
identified as associated with prognosis. Such methods wherein an internal
control is useful include for example quantitative polymerase chain
reaction (PCR) or fluorescent in situ hybridization (FISH). Optionally,
the copy number can be compared to the centromere for example when using
FISH. Typically a normal or control genomic profile refers to a single
genomic copy on each of the two alleles. For example in the array-CGH,
the control is a normal reference genomic DNA that is assumed to have 2
copies of each gene. In other examples, a positive control is employed,
for example, a sample or standard corresponding to subject comprising the
gain or loss associated with prognosis and/or response to chemotherapy,
useful for example for quantitative PCR and/or FISH methods, for example
included in quantitative PCR and/or FISH based kits. Based on the
teachings herein and knowledge in the field, a person skilled in the art
would readily be able to identify suitable controls for the methods
described herein.

[0063] The term "disease free subject" refers to a subject that is free of
lung cancer.

[0064] The term "microarray" as used herein, refers to an array of
distinct polynucleotides or oligonucleotides synthesized or spotted (e.g.
in the case of BAC clones) on a substrate, such as paper, nylon or other
type of membrane, filter, chip, glass slide, or any other suitable solid
support.

[0065] The terms "complementary" or "complementarity", as used herein,
refer to the natural binding of polynucleotides under permissive salt and
temperature conditions by base-pairing. For example, the sequence "A-G-T"
binds to the complementary sequence "T-C-A". Complementarity between two
single-stranded molecules may be "partial", in which only some
nucleotides or portions of the nucleotide sequences of the nucleic acids
bind, or it may be complete when total complementarity exists between the
single stranded molecules. The degree of complementarity between nucleic
acid strands has significant effects on the efficiency and strength of
hybridization between nucleic acid strands.

[0066] "Amplification of polynucleotides" can be achieved by utilization
of s methods such as the polymerase chain reaction (PCR), including for
example quantitative PCR, multiplex PCR and multiplex ligation dependent
probe amplification (MLPA), ligation amplification (or ligase chain
reaction, LCR) and amplification methods based on the use of Q-beta
replicase. These methods are well known and widely practiced in the art.
Reagents and hardware for conducting PCR are commercially available.
Primers useful to amplify specific sequences from selected genomic
regions are preferably complementary to, and hybridize specifically to
sequences flanking the target genomic regions.

[0067] The term "reference profile" as used herein refers to a reference
expression profile, a reference genomic profile, and/or a reference gene
copy number profile according to the context.

[0068] A "reference expression profile" as used herein refers to the
expression signature of a subset of biomarkers (e.g. one or more), which
correspond to genes associated with a prognosis class e.g. poor prognosis
or good prognosis +/- chemotherapy and/or a control.

[0069] The term "expression level" as used herein refers to the absolute
or relative amount of the transcription and/or translation product of a
gene described herein and includes RNA and polypeptide products.

[0070] A "reference gene copy number profile" as used herein refers to the
gene copy number of a subset of genes (e.g one or more) listed in Tables
5, 6, 7, 8, 9 and/or 11. The reference gene copy number profile is
optionally a reference number, typically 2, and/or identified using for
example using normal human tissue and/or cells and/or tissue and/or cells
from lung cancer. Normal tissue and/or cells includes for example, tumor
adjacent non-neoplastic tissue and/or cells and/or tissue and/or cells
from a lung cancer disease free subject. The reference gene copy number
profile is accordingly a reference signature of the copy number of a
subset of genes in Tables 5, 6, 7, 8, 9 and/or 11, to which the subject
gene copy number of the corresponding genes in a sample of a subject are
compared.

[0071] The term "genomic profile" as used herein refers to the genomic
structural signature of a subject genome. A number of variations and
alterations referred to as copy number variations, have been
characterized including amplifications and deletions, a subset of which
are associated with disease. The alterations can comprise small and large
amplifications and/or deletions which can occur through out the genome.

[0072] The phrase "determining a genomic profile" as used herein refers to
detecting the presence, absence, frequency, variability and/or length of
one or more genomic alterations including amplifications and deletions of
all or part of one or more MCRs and/or which may or may not comprise
alterations in the coding nucleic acid sequence of genes e.g., can
comprise alterations in the intergenic regions of the genome, such as
those found for example on 12q, 8q and 11q. Genomic alterations
comprising amplifications and deletions in all or part of one or more
genes comprise those listed in Tables 5, 6, 7, 8, 9 and/or 11. A person
skilled in the art will appreciate that a number of methods can be used
to determine a genomic profile, including for example fluorescence and
other non-fluorescent types of in situ hybridization (FISH, CISH or
others), and quantitative PCR (qPCR), multiplex PCR including for example
multiplex ligation dependent probe amplification (MLPA) and array CGH.

[0073] The term "reference genomic profile" as used herein refers a
genomic signature comprising genomic alterations, associated with
prognosis with or without chemotherapy. The reference genomic profile is
optionally a normal reference genomic DNA (e.g. a control) that is
assumed to have 2 copies of each gene and/or is derived from normal human
tissues and/or cells. The reference genomic profile is accordingly for
example, normal genomic copy number to which a subject genomic profile is
compared for classifying the tumor or determining or predicting clinical
outcome.

[0074] The term "chemotherapy" as used herein means treatment with
anticancer drugs, including but not limited to treatment with vinca
alkaloids for example vinorelbine vinblastine, vincristine, vinflunine
and/or vindesine in for example IV or oral form and/or platinum analogues
for example cisplatin, carboplatin, paraplatin, satraplatin and/or
oxaliplatin in for example IV or oral form.

[0075] The term "chemotherapeutic" as used herein means an anticancer
drug, including but not limited to treatment with mitotic inhibitors such
as vinca alkaloids for example vinorelbine vinblastine, vincristine,
and/or vindesine or analogs thereof and/or DNA alkylating agents such as
platinum based chemotherapeutics for example cisplatin, carboplatin and
oxaliplatin.

[0076] The term "similar" or "similarity" as used herein with respect to a
reference profile refers to similarly in both the identity and quantum of
change in expression level of a biomarker, genomic alteration, or gene
copy number variation compared to a control where the control is for
example derived from a normal cell and/or tissue or has a known outcome
class such as poor survival or good survival.

[0077] The term "similarity in expression" as used herein means that there
is no or little difference, for example no statistical difference, in the
level of expression of the biomarkers between the test sample and the
control and/or between good and poor prognosis groups defined by
biomarker expression levels.

[0078] The term "most similar" in the context of a reference profile
refers to a reference profile that is associated with a clinical outcome
that shows the greatest number of identities and/or degree of changes
with the subject profile.

[0079] The term "differentially expressed" or "differential expression" as
used herein refers to biomarkers described herein that are expressed at
one level in a prognostic group and expressed at another level in a
control. The differential expression can be assayed by measuring the
level of expression of the transcription and/or translation products of
the biomarkers, such as the difference in level of messenger RNA
transcript expressed or polypeptide expressed in a test sample and a
control. The difference can be statistically significant.

[0080] The term "difference in the level of expression" refers to an
increase or decrease in the measurable expression level of a given
biomarker expression product as measured by the amount of messenger RNA
transcript and/or the amount of polypeptide in a sample as compared with
the measurable expression level of a given biomarker in a control. In one
embodiment, the differential expression can be compared using the ratio
of the level of expression of a given biomarker or biomarkers as compared
with the expression level of the given biomarker or biomarkers of a
control, wherein the ratio is not equal to 1.0. For example, an RNA or
polypeptide is differentially expressed if the ratio of the level of
expression in a first sample as compared with a second sample is greater
than or less than 1.0. For example, a ratio of greater than 1.1, 1.2,
1.5, 1.7, 2, 3, 3, 5, 10, 15, 20 or more, or a ratio less than 0.9, 0.8,
0.6, 0.4, 0.2, 0.1, 0.05, 0.001 or less. A sample can be compared to a
group to identify differential expression. For example, one could compare
a sample of interest to a group of control samples and use a p-value to
demonstrate statistically that the sample of interest is for example
overexpressing the RNA product of a gene or has an increased DNA copy
number at that gene compared to control samples.

[0081] The term "prognosis" as used herein refers to a clinical outcome
e.g. a poor survival or a good survival, and includes for example
survival outcome in the absence of chemotherapy and/or improved survival
with administration of chemotherapy. Good prognosis and improved survival
are used herein interchangeably as are poor prognosis and poor survival.
As demonstrated herein, prognosis is associated with the presence or
absence of a gain or loss of specific MCRs and genes described herein,
compared to a reference profile such as a reference expression profile,
or a reference gene copy number profile of a suitable comparator group.
For example, subjects with gains in MCRs and/or genes listed in for
example Tables 1, 2, 5, 9, 10 and/or 11 or loss of MCRs and/or genes in
Table 3, 4, and/or 7 have a poor prognosis or poor survival compared to
subjects not having these gains or losses for regions identified.
Accordingly, the prognosis provides an indication of disease progression
and includes an indication of likelihood of recurrence, metastasis, death
due to disease e.g. survival, tumor subtype or tumor type.

[0082] The term "associated with a prognosis" as used herein refers to
gains and/or losses in all or part of a MCR and/or gene associated with
survival identified in the Tables as associated with for example, poor
survival in the absence of chemotherapy and/or listed in the Tables as
associated with improved survival with chemotherapy, as well as for
example MCRs and/or genes listed in the Tables as associated with good
and/or prognosis. The term "associated with a poor prognosis" identifies
the subset shown to statistically or trend to poor survival with surgery
alone e.g. in the absence of chemotherapy (and/or the presence of
chemotherapy for gains at AK024870 and/or CPSF6).

[0083] The term "tumour responsiveness" as used herein refers to the
likelihood that a subject's lung cancer will or will not respond to
chemotherapy treatment. It has been determined that a subset gains or
losses associated with prognosis are associated with benefit from
chemotherapy such that a subject with these gains or losses have an
improved survival when treated with chemotherapy compared to a subject
not receiving chemotherapy with the same gain or loss. Gains have also
been associated with worse survival. For example, a gain or increased
expression of ANK024870 and/or CPSF6 is associated with worse survival
with administration of chemotherapy.

[0084] The term "classifying" as used herein refers to assigning, to a
class or kind, an unclassified item. A "class" or "group" then being a
grouping of items, based on one or more characteristics, attributes,
properties, qualities, effects, parameters, etc., which they have in
common, for the purpose of classifying them according to an established
system or scheme. For example, subjects having gains associated with poor
prognosis, such as gains in MCRs and/or genes listed in for example
Tables 1, 2, 5, 9, 10 and/or 11, or losses of MCRs and/or genes listed in
Table 3, 4 and/or 7, define a class with poor prognosis. Also for
example, subjects having a gain in a Table 5, 9 or 11 gene or loss in a
Table 7 gene identified as showing benefit from receiving chemotherapy,
define a class that benefit from receiving chemotherapy. Similarly,
subjects for example with a gain in a Table 6 gene or a loss of a Table 8
gene define a class with good prognosis.

[0085] The term "loss" or "gain" refers with respect to a genomic profile
refers to a change in copy number, for example the loss can be on the
plus strand or the minus strand and can involve loss of one or both
alleles. Similarly, a "gain" can for example be a gain on the plus strand
or the minus strand and can involve gain on one or both alleles. The gain
can additionally be the gain of 1 or more copies.

[0086] The term "high amplitude gain" or "high level amplification" as
used herein refers to a copy number variation of a MCR or gene
amplification where the average log 2 value, as assigned by DNAcopy
analysis, in the gained samples, was greater than 0.15. For example, high
amplification gains were identified as described in the Examples and
include for example MCRs listed in Table 10 and genes listed in Table 11.

[0087] The term "prognosing" as used herein means predicting clinical
outcome such as survival and/or response to chemotherapy for example by
identifying the class a subject belongs to according to the presence of a
gain or loss of a genomic region such as 12q, 11q, 8q, 1p, or 14q or a
region (MCR) or gene identified in any one of Tables 1 to 11. Where one
or more gains or losses are detected, clinical outcome can be based on a
subject's similarity to a control and/or a reference profile and/or
biomarker expression level associated with a prognosis. Methods of
prognosis described herein can optionally be included in multivariate
models incorporating known prognostic clinical factors, such as age, sex
stage and grade.

[0088] The term "good survival" as used herein refers to an increased
disease free survival for example as compared to subjects in a suitable
comparator "poor survival" group e.g. not having a gain or loss
associated with good prognosis or improved response to chemotherapy. The
term "poor survival" as used herein refers to an increased risk of death
and/or disease occurrence as compared to subjects in a suitable
comparator "good survival" group e.g. having a gain or loss associated
with good prognosis or improved response to chemotherapy. For example,
subjects comprising a gain or loss of a MCR or gene or altered biomarker
expression described herein as associated with poor prognosis, such as
genes and MCRs listed in Tables 1, 2, 5, 7, and/or 9-11, have a poor
survival compared to subjects not comprising such a loss, gain or altered
expression as indicated therein. As another example, subjects not
receiving chemotherapy who comprise a gain or loss associated with
improvement when treated with chemotherapy, for example such as MCRS
listed in Table 1, 2, and/or 3 and/or genes listed in Tables 5, 7, 9
and/or 11 associated with improvement with chemotherapy, have poor
survival when not treated with chemotherapy compared to subjects with the
same gain, loss or altered expression who receive chemotherapy. As a
further example, subjects who comprise a gain or loss not associated with
improvement when treated with chemotherapy have a poor prognosis compared
to individuals without the gain, or loss. Similarly, for example, a good
survival group comprises subjects comprising a gain or loss or biomarker
expression described herein associated with good prognosis, for example a
gain or loss listed in Table 6 and/or 8 respectively. As another example,
subjects receiving chemotherapy that comprise gains or losses associated
with improved survival with chemotherapy, such as the particular MCRs
listed in Table 1, 2 and/or 3 and/or the genes in Tables 5, 7, 9 and/or
11 identified as associated with significant improvement with
chemotherapy have good survival when treated with chemotherapy compared
to subjects with the similar gain, loss or expression who do not receive
chemotherapy. Subjects in a good survival group or good survival group
when treated with chemotherapy are at less risk of death 5 years after
surgery. Subjects in a poor survival group or poor survival when not
treated with chemotherapy group are at greater risk of death within 5
years from surgery. For example a poor survival group comprises subjects
having a 5 year survival rate of less than 80%. As used herein, good
survival indicates good prognosis and poor survival indicates poor
prognosis.

[0089] The term "genes associated with good survival" or "genes associated
with good prognosis" as used herein refers to genes listed in Table 6,
for example RAB11FIP1 and genes listed in Table 8, for example, C6orf15,
CDYL, HLA-DOA, KIFC1, MSH5/C6orf26, NCR3, RXRB, and/or TCL6.

[0090] The term "MCRs associated with good survival" or "MCRs associated
with good prognosis" as used herein refer to MCRs associated with good
prognosis for example the MCRs comprising the genes listed in Tables 6
and/or 8.

[0093] The term "genes not associated with improvement when treated with
chemotherapy" as used herein refers to genes for example listed in Table
5 identified as not showing significant improvement when treated with
chemotherapy, for example ADCY2, (clone Z146), ANKH, CDH18, OXCT1, UTRN,
cDNA DKFZp434E2423, C9orf68, ITGA7, CDK2/BCDO2, ERBB3, DLST/PA2G4, PRIM1,
TRHDE, OR1E1/OR1E2, and/or RCVRN; and/or genes listed in Table 7
identified as not showing significant improvement when treated with
chemotherapy, for example AHCYL1, ATP1A1, IGSF3, ELF1, RGC32, ESD, TAF1C,
and/or MAP1LC3B, as well as genes listed in Table 9 and/or 11 so
identified. Detection of these genes for example is useful for selecting
a treatment regimen. For example since subjects comprising losses or
gains at these loci do not demonstrate improved prognosis with cisplatin,
and/or venolrebine, chemotherapeutics that are not related to cisplatin
and/or venolrebine e.g. a different class of drug, may be indicated.

[0094] The term "minimal common region" or "MCR" refers to the a region
determined to be commonly gained or lost in subjects belonging to a
particular class such as good prognosis when treated chemotherapy. A
subject may have a gain or loss that comprises the MCR and/or comprises a
portion of the MCR. For example the minimal common regions associated
with poor prognosis in the absence of chemotherapy, and/or improved
prognosis upon treatment with chemotherapy, are listed in Tables 1-11.
The MCR start and stop positions refer to positions in NCBI human genome
build 36.3 which corresponds to hg18.

[0095] As used herein, "treatment" or "treating" is an indicated approach
for obtaining beneficial or desired results, including clinical results,
for example an indicated approach for lung cancer. Beneficial or desired
clinical results can include, but are not limited to, alleviation or
amelioration of one or more symptoms or conditions, diminishment of
extent of disease, stabilized (i.e. not worsening) state of disease,
preventing spread of disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, prolonging survival as
compared to expected survival if not receiving treatment and remission
(whether partial or total), whether detectable or undetectable. For
example surgery is indicated for Stage I lung cancers, and surgery plus
adjuvant chemotherapy is indicated for subjects with more advanced
stages. The methods described herein are useful for example, for
identifying subjects with lung cancer that benefit from receiving
chemotherapy.

[0096] The phrase "selecting a treatment" as used herein refers to
selecting a chemotherapeutic regimen, for example a regimen comprising a
platinum based chemotherapeutic such as cisplatin, a regimen comprising a
vinca alkyloid such as vinolrebine or a treatment regimen comprising a
combination thereof, that is useful for obtaining beneficial results such
as prolonging survival. Alternatively for example, where MCRs or genes
that are not associated with improvement with chemotherapy or good
prognosis, the treatment selected is a regimen that does not comprise a
platinum based chemotherapeutic such as cisplatin, a regimen comprising a
vinca alkyloid such as vinolrebine or a treatment regimen comprising a
combination thereof.

[0097] The term "subject" such as a "subject" to be diagnosed, prognosed,
staged, screened, assessed for risk, subject for selection of a
treatment, and/or treated by the subject methods and articles of
manufacture can mean either a human or non-human animal, preferably a
human being.

[0098] The term "sample", "test sample" or "biological sample" as used
herein refers to any fluid, cell or tissue sample from a subject which
can be assayed for genomic alterations or biomarker expression products
e.g. for determining a genomic profile or an expression profile,
depending on the method and comprises without limitation lung tumor
tissue and/or cells, derived from, for example, lung biopsy, for example
obtained by bronchoscopy, needle aspiration, thoracentesis and/or
thoracotomy, and/or derived from cells found in sputum. The term could
also be used for example to refer to metastatic tumour tissue obtained
from the brain or liver or other site.

[0099] The phrase "determining the expression level of biomarkers" as used
herein refers to determining or quantifying RNA and/or polypeptides
expressed by the biomarkers. The term "RNA" includes mRNA transcripts,
and/or specific spliced variants of mRNA. The term "RNA product of the
biomarker" as used herein refers to RNA transcripts transcribed from the
biomarkers and/or specific spliced variants. In the case of
"polypeptide", it refers to polypeptides translated from the RNA
transcripts transcribed from the biomarkers. The term "polypeptide
product of the biomarker" refers to polypeptide translated from RNA
products of the biomarkers.

[0100] The term "nucleic acid" as used herein refers to a polynucleotide
molecule and includes DNA and RNA and can be either double stranded or
single stranded. The nucleic acid molecules contemplated by the present
disclosure include isolated nucleotide molecules which hybridize
specifically to genomic DNA, RNA product of a biomarker, polynucleotides
which are complementary to a RNA product of a biomarker of the present
disclosure, nucleotide molecules which act as probes, or nucleotide
molecules which are specific primers for a MCR or gene gained or lost set
out in Tables 1-11, including for example the probes and primers listed
in Tables 12 and 13.

[0101] The term "isolated nucleic acid" as used herein refers to a nucleic
acid substantially free of cellular material or culture medium when
produced by recombinant DNA techniques, or chemical precursors, or other
chemicals when chemically synthesized. An "isolated nucleic acid" is also
substantially free of nucleotides which naturally flank the nucleic acid
(i.e. nucleotides located at the 5' and 3' ends of the nucleic acid) from
which the nucleic acid is derived.

[0102] The term "hybridize" refers to the sequence specific non-covalent
binding interaction with a complementary nucleic acid. In a preferred
embodiment, the hybridization is under high stringency conditions.
Appropriate stringency conditions which promote hybridization are known
to those skilled in the art, or can be found in Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. For
example, 6.0× sodium chloride/sodium citrate (SSC) at about
45° C., followed by a wash of 2.0×SSC at 50° C. may
be employed when hybridization is detecting expression levels, for
example by northern or slot blot analysis. For array CGH, hybridization
often occurs with labeled DNA for patient and reference DNA added to a
solution including formamide and SSC (2.0×). The DNA/hybridization
buffer mixture is allowed to competitively hybridize at 45° C. to
the array (and its targets) for ˜36-40 hours, after which washes
take place. Signal intensities at each arrayed element are then
evaluated. A detailed description of array CGH hybridization protocols is
provided in Buys et al., "Key Features of Bacterial Artificial Chromosome
Microarray Production and Use" in DNA Microarrays (Methods Express
Series) (Schena M, ed.), Scion Publishing, Ltd. Bloxham, Oxfordshire, UK,
pp. 115-145 (ISBN: 9781904842156) (please see section 2.5 in particular).

[0103] The term "primer" as used herein refers to a nucleic acid sequence,
whether occurring naturally as in a purified restriction digest or
produced synthetically, which is capable of acting as a point of
synthesis of when placed under conditions in which synthesis of a primer
extension product, which is complementary to a nucleic acid strand is
induced (e.g. in the presence of nucleotides and an inducing agent such
as DNA polymerase and at a suitable temperature and pH). The primer must
be sufficiently long to prime the synthesis of the desired extension
product in the presence of the inducing agent. The exact length of the
primer will depend upon factors, including temperature, sequences of the
primer and the methods used. A primer typically contains 15-25 or more
nucleotides, although it can contain less. The factors involved in
determining the appropriate length of primer are readily known to one of
ordinary skill in the art.

[0104] The term "primer pair" as used herein refers a set of primers which
can produce a double stranded nucleic acid product complementary to a
portion of the RNA products of the biomarker or sequences complementary
thereof.

[0105] The term "probe" and/or "hybridization probe" as used herein refers
to a nucleic acid sequence that will hybridize to a nucleic acid target
sequence, for example. For example, the probe hybridizes to a RNA product
of the biomarker or a nucleic acid sequence complementary thereof for
detecting gene expression or hybridizes a genomic region comprising a
gain or loss of a genomic region described herein associated with
prognosis. The length of probe depends on the hybridization conditions
and the sequences of the probe and nucleic acid target sequence. For
example, the probe comprises at least 8, 10, 15, 20, 25, 50, 75, 100,
150, 200, 250, 400, 500 or more nucleotides in length, for example
complementary to at least 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250,
400, or 500 contiguous nucleotides of a gene listed in Table 5, 6, 7, 8,
9 and/or 11, or a genomic region alteration such as a MCR and/or region
flanking a MCR described herein, for example in Tables 1 to 11, for
example Table 1, 2, 3, 4 and/or 10. The probe can further be 90%, 95, 96,
97, 98, 99, 99.5, 99.9% identical to the at least 8, 10, 15, 20, 25, 50,
75, 100, 150, 200, 250, 400, or 500 contiguous nucleotides of a gene
listed in Table 5, 6, 7, 8, 9 and/or 11, or a genomic region alteration
such as a MCR and/or region flanking a MCR described herein, for example
in Table 1, 2, 3, 4 and/or 10. The probe can also for example comprise a
MCR or a gene associated with prognosis. For example the probe can be a
bacterial artificial chromosome (BAC) clones and can comprise the target
sequence as well as additional sequence. In this case, the probe can be
at least 50 000, 100 000, 150 000 and/or 200 000 nucleotides, for example
150 000-200 000 base pairs The probe can for example comprised in an
array, for example, on a solid support, for example array for CGH. For
example, BAC clone probes on the array are usually in the 150,000-200,000
bp range. Labelled DNA and reference DNA generated from subject and
reference DNA samples are typically a few hundred by in size (small
fragments may be excluded after labeling or during washing steps). These
subject DNA and reference DNA are generated for example, using a random
priming reaction, such that their lengths will vary. See for example Buys
et al. reference (above) and citations within (e.g. original citation at
Feinberg & Vogelstein Anal. Biochem, 132, 6-13.)

[0106] The term "antibody" as used herein is intended to include
monoclonal antibodies, polyclonal antibodies, and chimeric antibodies.
The antibody may be from recombinant sources and/or produced in
transgenic animals. The term "antibody fragment" as used herein is
intended to include Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers,
minibodies, diabodies, and multimers thereof and bispecific antibody
fragments. Antibodies can be fragmented using conventional techniques.
For example, F(ab')2 fragments can be generated by treating the antibody
with pepsin. The resulting F(ab')2 fragment can be treated to reduce
disulfide bridges to produce Fab' fragments. Papain digestion can lead to
the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv,
ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and
other fragments can also be synthesized by recombinant techniques.

[0107] The term "biomarker" as used herein refers to a gene that is
altered in its gene copy number in a poor prognosis class and/or a good
prognosis class e.g. with or without chemotherapy, compared to a control
and/or is differentially expressed in subjects in poor and good prognosis
classes. For example the term "biomarkers" includes one or more of the
genes listed in Table 5, 6, 7, 8, 9 and/or 11.

[0108] The definitions and embodiments described in particular sections
are intended to be applicable to other embodiments herein described for
which they are suitable as would be understood by a person skilled in the
art.

II. Methods

[0109] Lung cancer remains the leading cause of cancer death in Canada
with an overall 5-yr survival rate of 16%. Up to 40% of lung cancer
patients are potentially curable by surgery, yet their risk of dying from
the disease remains high at 50%. Post-surgery chemotherapy is a toxic
therapy but may improve cure rate. New methods of classifying lung
cancers are needed for making more informed decisions on chemotherapy,
based on specific molecular markers present in each cancer. Using a CGH
microarray, small regions of chromosomes have been identified that when
gained or lost in lung cancers, impart a worse prognosis with surgery
alone, and a subset of these also show a significant benefit with current
standard chemotherapy. After testing individual genes within these
regions by quantitative polymerase chain reaction, DNA copy number gains
located on 1p, 8q, 11q, 12q, and 14q were confirmed to impart a worse
prognosis in the absence of chemotherapy, and/or an improved response to
chemotherapy.

[0110] Accordingly in an aspect, the disclosure provides a method for
determining a lung cancer prognosis in a subject, the method comprising:
determining a genomic profile comprising detecting one or more genomic
alterations in chromosomes 2, 11, 4, 5, 7, 9, 12, 17, 19, 20, 8, 1, 13,
16, 6 and/or 14 listed in Tables 1-11 in a biological sample from the
subject; wherein the prognosis is determined to be poor in the absence of
chemotherapy when the genomic profile comprises a gain of one or more
minimal common regions (MCRs) or genes within chromosomes 1, 2, 11, 12,
4, 5, 6, 7, 9, 12, 14, 16, 17, 19 and 20 listed as associated with poor
prognosis in Tables 1, 2, 5, 9, 10, and 11, and/or a loss of one or more
MCRs or genes within chromosomes 1, 5, 8, 13 and/or 16 listed as
associated with poor prognosis in Tables 3 and/or 7 and the prognosis is
determined to be good in the absence of chemotherapy when the genomic
profile comprises a genomic gain of an MCR or gene within chromosome 8
listed as associated with good prognosis in Table 6 and/or a loss of one
or more MCRs or genes within chromosome 2, 6, 9 or 14 listed as
associated with good prognosis in Table 8 relative to the control.

[0111] In an embodiment, the method comprises: (a) determining a genomic
profile comprising detecting one or more genomic alterations in
chromosomes 2, 11, 4, 5, 7, 9, 12, 17, 19, 20, 8, 1, 13, 16, 6 and/or 14
listed in Tables 1-11 in a biological sample from the subject; (b)
determining the lung cancer prognosis for the subject by comparing the
genomic profile with one or more controls, wherein the prognosis is
determined to be poor when the genomic profile comprises a gain of one or
more minimal common regions (MCRs) or genes within chromosomes 1, 2, 11,
12, 4, 5, 6, 7, 9, 12, 14, 16, 17, 19 and 20 listed as associated with
poor prognosis in Tables 1, 2, 5, 9, 10, and 11, and/or a loss of one or
more MCRs or genes within chromosomes 1, 5, 8, 13 and/or 16 listed as
associated with poor prognosis in Tables 3 and/or 7 and the prognosis is
determined to be good when the genomic profile comprises a genomic gain
of an MCR or gene within chromosome 8 listed as associated with good
prognosis in Table 6 and/or a loss of one or more MCRs or genes within
chromosome 2, 6, 9 or 14 listed as associated with good prognosis in
Tables 6 and/or 8 relative to the control.

[0112] In an embodiment, the method comprises obtaining a biological
sample for determining the genomic profile.

[0113] In another embodiment, the disclosure provides a method for
determining a lung cancer prognosis in a subject, the method comprising:
detecting the presence of a genomic alteration at a locus identified in
Tables 1-11 in a biological sample from the subject, wherein the
prognosis is determined to be poor in the absence of chemotherapy when a
gain of a MCR or gene listed in Tables 1, 2, 5, 9, 10 and/or 11 and/or a
loss of a MCR or gene listed in Table 3 and/or 7 is detected; and the
prognosis is determined to be good when a gain of a MCR or gene listed in
Table 6 and/or loss of a MCR or gene in Table 4 and/or 8 is detected
relative to a control.

[0114] In an embodiment, the genomic alteration detected comprises a gain
or loss of DNA copy number at an MCR listed in Tables 1-11, for example
Table 1, 2, 3, 4 and/or 10. In another embodiment, the presence or
absence of a gain of DNA copy number is detected at an MCR at 1p at or
within basepair positions 41265460 to 43221579. In another embodiment,
the presence or absence of a gain of DNA copy number is detected at an
MCR at 8q at or within basepair positions 128289292 to 128936748. In
another embodiment, the presence or absence of a gain of DNA copy number
is detected at an MCR at 11q at or within basepair positions 68572940 to
70388868. In another embodiment, the presence or absence of a gain of DNA
copy number is detected at an MCR at 14q at or within basepair positions
96994959 to about 99058653. In another embodiment, the presence or
absence of a gain of DNA copy number is detected at an MCR at 12q at or
within basepair positions 50731457 to 51457372. In another embodiment,
the presence or absence of a gain of DNA copy number is detected at an
MCR at 12q at or within basepair positions 52696908 to 53538441. In
another embodiment, the presence or absence of a gain of DNA copy number
is detected at an MCR at 12q at or within basepair positions 55933813 to
57461765. In another embodiment, the presence or absence of a gain of DNA
copy number is detected at an MCR at 12q at or within basepair positions
64438067 to 68503251. In another embodiment, the presence or absence of a
gain of DNA copy number is detected at an MCR at 14q at or within
basepair positions 96994959 to 99058653.

[0115] In another embodiment, the genomic alteration detected comprises
all or part of a MCR listed in Table 1, 2, 3, 4 and/or 10. In an
embodiment, the genomic alteration detected comprises all or part of a
MCR listed in Table 10.

[0116] In an embodiment, the method comprises determining a genomic
profile comprising detecting one or more genomic alterations listed Table
1, 2, 5, 9, 10 and/or 11, in a biological sample from the subject; (b)
determining the lung cancer prognosis for the subject by comparing the
genomic profile with one or more controls, wherein the prognosis is
determined to be poor in the absence of chemotherapy when the genomic
profile comprises a gain of one or more minimal common regions (MCRs) or
genes listed in Table 1, 2, 5, 9, 10 and/or 11.

[0117] In another embodiment, the method comprises determining a genomic
profile comprising detecting one or more genomic alterations in
chromosomes 1, 5, 8, 13 and 16 listed in Table 3 and/or 7 wherein the
prognosis is determined to be poor in the absence of chemotherapy when
the genomic profile comprises a loss of one or more MCRs or genes within
chromosomes 1, 5, 8, 13 and 16 listed in Table 3 and/or 7.

[0118] In a further embodiment, the method comprises determining a genomic
profile comprising detecting a genomic alteration or gene gain in
chromosome 8 listed as associated with good prognosis in Table 6, wherein
the prognosis is determined to be good when the genomic profile comprises
a gain of the MCR or the gene within chromosome 8 listed as associated
with good prognosis.

[0119] In another embodiment, the method comprises determining a genomic
profile comprising detecting one or more genomic alterations in
chromosomes 6 and/or 14, wherein the prognosis is determined to be good
when the genomic profile comprises a loss of one or more MCRs within
chromosomes 6 and/or 14 listed in Table 8.

[0120] In another aspect, all or part of genes located within the MCRs
gained or lost, for example the MCRs listed in any one of Tables 1 to 11,
for example, Tables 1, 2 and/or 10 are detected. Detection of an
increased or decreased DNA copy number of a gene (e.g. a gain,
amplification, or loss of said gene) comprised therein can be indicative
of the presence or absence of a gain, amplification, or loss at the
corresponding MCR. For example, DQ515898, DQ515897, and MYC genes are
found within the MCR at basepair positions 128289292 to 128936748 on
chromosome arm 8q, CCND1 and FGF3 genes are found within the MCR at
basepair positions 68572940 to 70388868 on chromosome arm 11q, and
B4GALNT1, OS9, CENTG1, CDK4, and TSFM are genes found within the MCR at
basepair positions 55933813 to 57461765 on chromosome arm 12q.

[0121] In an embodiment, the gene detected is selected from the group,
DQ515898, DQ515897, and MYC. In a further embodiment, the gene detected
is selected from the group consisting of AK024870, NUP107, MDM2, CPSF6,
and BCL11B. In a further embodiment, the gene detected is selected from
the group consisting of GUCA2A, PPIH, LEPRE1, CR623026, and C1orf50. In a
further embodiment, the gene detected is selected from the group
consisting of CCND1 and FGF3. In a further embodiment, the gene detected
is selected from the group consisting of B4GALNT1, OS9, CENTG1, CDK4, and
TSFM.

[0123] The MCRs described herein as associated with prognosis comprise
gains or losses of genes listed in Tables 5, 6, 7, 8, 9 and/or 11, and of
the genomic regions listed in Tables 1 to 11 and particularly Tables 1,
2, 3, 4 and/or 10. The gain or loss can be all or part of any one of
these genes. In an embodiment, the detected gain or loss comprises
amplification and/or deletion of the entire gene.

[0124] Accordingly, in a further embodiment, the prognosis is determined
to be poor, in the absence of chemotherapy, when the genomic profile
comprises a gain of a MCR comprising all or part of a gene listed in
Table 5, 9 and/or 11 associated with poor prognosis and/or comprises a
loss of a MCR comprising all or part of a gene listed in Table 7
associated with poor and/or comprises a gain of an MCR in table 1, 2
and/or 10 associated with poor prognosis, and the prognosis is determined
to be good, in the absence of chemotherapy, when the genomic profile
comprises a gain of a MCR comprising all or part of gene listed in Table
6 and/or a loss of a MCR comprising all or part of a gene listed in Table
8 relative to the control.

[0125] The genomic profile can be determined by various methods for
example by determining a hybridization pattern using a probe that
hybridizes to a region described herein as associated with a prognosis or
outcome. In an embodiment, a set of probes are used. In another
embodiment the probe is a chromosomal probe.

[0126] In an embodiment, detection of one of the gains losses described
herein is sufficient for association with prognosis and/or response to
chemotherapy.

[0127] In an embodiment, the method comprises hybridizing a chromosomal
probe or a set of chromosomal probes to the biological sample, and
detecting the presence or absence of hybridized probe.

[0128] In an embodiment, the probe is complementary to at least 8, 10, 15,
20, 25, 50, 75, 100, 150, 200, 250, 400, 500 contiguous nucleotides of a
gene listed in Table 5, 6, 7, 8, 9 and/or 11, or a genomic region
alteration such as a MCR and/or region flanking a MCR described herein,
for example in Table 1, 2, 3, 4 and/or 11. In another embodiment, the
probe is at least or greater than 90, 95, 96, 97, 98, 99, 99.5 or 99.9%
identical to a gene listed in Tables 5, 6, 7, 8, 9 and/or 11, or a region
in listed in any one of Tables 1-11, for example Tables 1, 2, 3, 4 and/or
10.] Alternatively, for example the probe can be a bacterial artificial
chromosome (BAC) clone and can comprise the target sequence. In this
case, the probe can be at least 50,000 bp, at least 100,000 bp, at least
150,000 bp and/or at least 200 000 bp, for example 150 000-200 000 bp The
probe can for example comprised in an array, for example, on a solid
support. Accordingly, in another embodiment, the set of chromosomal
probes is comprised in an array.

[0129] In a further embodiment, the probes are labeled, for example the
probes are fluorescently labeled. In another embodiment, the subject DNA
and the reference DNA is labeled.

[0130] Accordingly in another embodiment, the method comprises: (a)
determining a hybridization pattern of a chromosomal probe in a
biological sample from the subject, wherein the probe hybridizes to a
chromosome selected from the group 11, 4, 5, 6, 7, 9, 12, 17, 20, 8, 1,
13, 16, and/or 14 and (b) determining the lung cancer prognosis for the
subject based on the hybridization pattern, wherein the prognosis is
determined to be poor when the hybridization pattern indicates a gain of
one or more MCRs or genes within chromosome for example 1, 2, 8, 11, 12,
14 and/or 20 listed in Table 1, 2 and/or 10, or for example within
chromosome 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 14, 17, and 20 listed in Table
5, 9 and/or 11 and/or a loss of one or more MCRs or genes within
chromosomes 1, 13 and 16 listed in Table 7 and the prognosis is
determined to be good when the hybridization profile indicates a gain of
a MCR or gene within chromosome 8 in Table 6 and/or a loss of one or more
MCRs or genes within chromosome 6 or 14 in Table 8, relative to the
control.

[0131] Accordingly in another embodiment, the method comprises: (a)
determining a hybridization pattern of a chromosomal probe or set of
chromosomal probes in a biological sample from the subject, wherein the
set comprises one or more probes directed to one or more MCRs and/or
genes in chromosomes 2, 11, 4, 5, 6, 7, 9, 12, 14, 16, 17, 19, 20, 8, 1,
13, 16, 6 and/or 14 listed in Tables 1-11; and (b) determining the lung
cancer prognosis for the subject based on the hybridization pattern,
wherein the prognosis is determined to be poor when the hybridization
pattern indicates a gain or loss of one or more MCRs or genes associated
with poor prognosis and the prognosis is determined to be good when the
hybridization profile indicates a gain or loss of one or more MCRs or
genes associated with good prognosis relative to the control.

[0132] In an embodiment, the prognosis is determined to be poor when the
hybridization pattern indicates a gain of one or more MCRs or genes
listed in Table 1, 2, 5, 9 and/or 11 and/or a loss of one or more MCRs or
genes listed in Table 3 and/or 7. In an embodiment, the gain comprises
all or part of a gene listed in Table 5. In another embodiment, the gain
comprises all or part of a gene listed in Table 9. In yet another
embodiment, the gain comprises all or part of a gene listed in Table 11.
In another embodiment, the loss comprises all or part of a gene listed in
Table 7.

[0133] In yet a further embodiment, the prognosis is determined to be good
when the hybridization pattern indicates a gain of a MCR or gene within
chromosome 8 and/or a loss of one or more MCRs or genes within chromosome
6 or 14 relative to the control. In an embodiment, the gain comprises all
or part of RAB11FIP1. In another embodiment, the loss comprises all or
part of a gene listed in Table 8.

[0134] It has also been determined that subjects with a gain or loss of a
subset of MCRs or genes are associated with significant improvement in
survival and/or have improved tumor responsiveness with chemotherapy
compared to subjects with the gain or loss not treated with chemotherapy.

[0135] In another aspect, the disclosure includes a method for determining
a likelihood of improved survival or response with chemotherapy treatment
comprising detecting a gain of all or part of a MCR or gene listed in
Tables 1, 2, 5, 9, 10 and/or 11 associated with improved response to
chemotherapy, wherein a gain indicates the subject has a good prognosis
when treated with chemotherapy relative to a subject not treated with
chemotherapy.

[0136] In another aspect, the disclosure includes a method for determining
tumour responsiveness to a chemotherapy treatment comprising detecting a
gain of all or part of one or more of the genes listed in Tables 1, 2, 5,
9 or 11 associated with improved response to chemotherapy, wherein a gain
indicates the tumour is likely responsive to treatment with chemotherapy
relative to a tumour not comprising the gain.

[0137] Accordingly in an embodiment, a gain detected of all or part of one
or more of the following genes: MFSD7, D4S234E, ACOX3, SRD5A1, AQP2,
ACCN2, SLC11A2, SCN8A, KRT81, KRT1, ESPL1, NPFF, ATP5G2, HOXC11, NEUROD4,
ZBTB39, KIAA0286, INHBE, MARS, B4GALNT1, TSFM, DNMT3B and/or the loss of
all or part of one of the following genes: RHOC, ATP2C2, ZDHHC7, COC4I1,
FOXF1, indicates the subject has a good prognosis when treated with
chemotherapy relative to a subject not treated with chemotherapy.

[0138] In another embodiment, the gain associated with improved survival
with chemotherapy or improved tumor responsiveness is a gain of all or
part of one or more of the following genes: BAALC, ANGPT1, MYC, WISP1,
KRT81, KRT1, NEUROD4, and/or PA2G4 (e.g. Table 9 genes associated with
improved response to chemotherapy). In a further embodiment, the gain
associated with improved survival with chemotherapy or improved tumor
responsiveness is a gain of all or part of one or more of the following
genes: GUCA2A, PPIH, LEPRE1, CR623026, C1orf50, DQ515898, DQ515897, MYC
FGF3, KRT81, KRT1, FAM112B, B4GALNT1, CENTG1, BCL11B (e.g. Table 11 genes
associated with improved response to chemotherapy).

[0139] Another aspect provides a method of determining a lung cancer
prognosis in a subject, the method comprising detecting the presence of a
MCR and/or gene associated with improvement with chemotherapy, for
example a MCR of Table 1, 2, and/or 3, or a gene from Table 5 or 7,
wherein the gain or loss of a MCR and/or gene associated with improvement
with chemotherapy (as indicated in the relevant table) is indicative the
subject will have good prognosis relevant to a control, for example a
subject with the gain or loss not receiving chemotherapy.

[0140] In another aspect, the disclosure includes a method for determining
a likelihood of improved survival with chemotherapy treatment comprising
detecting a loss of all or part of a MCR or gene listed in Tables 3, 4, 7
and/or 8 associated with improved response to chemotherapy, wherein the
loss indicates the subject has a good prognosis when treated with
chemotherapy relative to a subject not treated with chemotherapy.

[0141] In another aspect, the disclosure includes a method for determining
tumour responsiveness to a chemotherapy treatment comprising detecting a
loss of all or part of a MCR or gene listed in Tables 3, 4, 7 and/or 8
associated with improved response to chemotherapy, wherein the loss
indicates the tumour is likely responsive to treatment with chemotherapy
relative to a tumour not comprising the loss.

[0142] In an embodiment, the chemotherapy comprises a platinum based
chemotherapeutic. In another embodiment, the chemotherapy comprises a
vinca alkaloid. In a further embodiment, the chemotherapy regimen
includes both a platinum based chemotherapeutic and a vinca alkyloid.

[0143] Expression data of the genes herein identified associated with
prognosis is also predicted to be useful for predicting prognosis.
Generally, with increasing gene dosage, gene expression levels would be
expected to increase. Similarly, with decreasing gene dosage, gene
expression would be expected to decrease. This is for example often the
case with heterozygous gene knock out in mice, and/or transgene copy
number in transgenic mice. For example, increased expression of a gene
whose gain is associated with poor outcome is expected to be indicative
of poor outcome and decreased expression of a gene, loss of which is
associated with poor outcome is expected to be indicative of poor
outcome. Similarly, increased expression of a gene, gain of which is
associated with good outcome is expected to be indicative of good outcome
and decreased expression of a gene, loss of which is associated with good
outcome, is expected to be indicative a good outcome. Gene expression can
be determined alone and/or in conjunction with genomic alterations.

[0144] Accordingly, another aspect provides a method for determining a
lung cancer prognosis in a subject, the method comprising: (a)
determining an expression profile comprising detecting an expression
level of one or more genes listed in Tables 5, 6, 7, 8, 9 and/or 11
associated with prognosis in a biological sample from the subject;
wherein the prognosis is determined to be poor when the expression
profile comprises a increased level of expression of one or more genes in
Table 5, 9 and/or 11 associated with poor prognosis and/or a decreased
expression in one or more genes listed in Table 7 and the prognosis is
determined to be good when the expression profile comprises increased
expression of RAB11FIP1 and/or decreased expression of one or more genes
in Table 8, relative to a control.

[0145] In an embodiment, the method includes step (b), said step (b)
comprising determining the lung cancer prognosis for the subject by
comparing the expression profile with one or more controls.

[0146] The expression level is optionally determined in addition to the
genomic copy number. Accordingly, in addition to determining the genomic
profile and/or the detecting the gain or loss of a MCR comprising all or
part of one or more genes listed in Tables 5, 6, 7, 8, 9 and/or 11, the
method further comprises detecting the expression level of a gene listed
in Table 5, 6, 7, 8, 9 and/or 11. In an embodiment, the expression level
of the gene all or partly gained or lost, is increased or decreased
respectively, relative to a control expression level.

[0147] In an embodiment, the expression level is detected using a probe
that binds a gene listed in Tables 5, 6, 7, 8, 9 and/or 11. In an
embodiment, the probe comprises at least 8, 10, 15, 20, 25, 50, 75, 100,
150, 200, 250, 400, 500 contiguous nucleotides complementary to a gene
listed in Table 5, 6, 7, 8, 9 and/or 11, or a gene with at least 90, 95,
98, 99, 99.5 or 99.9% identity to a gene in Table 5, 6, 7, 8, 9 and/or
11. The probe can for example be comprised in an array, for example, on a
solid support, for example array. In another embodiment, the expression
level is detected by detecting the presence or absence of hybridized
probe.

[0148] In a further embodiment, the probes are comprised in an array, for
example on a solid support. In another embodiment, the probes are labeled
or for example fluorescently labeled.

[0149] As described herein and mentioned above, prognostic associations
have been found for MCRs of gain located on 12q and 14q (e.g. Table 1 or
10). Such MCR gains were found by array-CGH and qPCR studies to be
significantly associated with poor survival in the absence of
chemotherapy. Predictive associations have been found for MCRs of gain
located on 1p, 8q, 11q, 12q, and 14q. Subjects with these MCRs were found
to have improved survival when treated with chemotherapy.

[0150] Accordingly, another aspect provides a method for determining a
lung cancer prognosis in a subject, the method comprising: (a)
determining a hybridization pattern of a chromosomal probe in a
biological sample from the subject, wherein the set comprises a probe to
the 6 Mb region of chromosome 12q, 8q or 11q; and (b) determining the
lung cancer prognosis for the subject based on the hybridization pattern,
wherein the prognosis is determined to be poor without chemotherapy when
the hybridization pattern indicates a gain of a MCR within the 6 Mb
region of chromosome 12q relative to a control and/or the prognosis is
determined to be good when treated with chemotherapy when the
hybridization pattern indicates a gain of a MCR within 8q and/or 11q.

[0151] In an embodiment, gain of DNA copy number at an MCR located on 1p
within basepair positions 41265460 to 43221579 is indicative of a good
prognosis with chemotherapy.

[0152] In another embodiment, gain of DNA copy number at an MCR within
basepair positions 128289292 to about 128936748 on the long arm of
chromosome 8 is indicative of a good prognosis with chemotherapy.

[0153] In another embodiment, gain of DNA copy number at an MCR within
basepair positions 68572940 to about 70388868 on the long arm of
chromosome 11 is indicative of a good prognosis with chemotherapy.

[0154] In another embodiment, gain of DNA copy number at an MCR within
basepair positions 50731457 to about 51457372 on the long arm of
chromosome 12 is indicative of a good prognosis with chemotherapy.

[0155] In another embodiment, gain of DNA copy number at an MCR within
basepair positions 52696908 to about 53538441 on the long arm of
chromosome 12 is indicative of a good prognosis with chemotherapy.

[0156] In another embodiment, gain of DNA copy number at an MCR within
basepair positions 55933813 to about 57461765 on the long arm of
chromosome 12 is indicative of a good prognosis with chemotherapy.

[0157] In another embodiment, gain of DNA copy number at an MCR within
basepair positions 96994959 to about 99058653 on the long arm of
chromosome 14 is indicative of a good prognosis with chemotherapy.

[0158] Several genes comprised within the 1p, 8q, 11q, 12q, and 14q MCRs
were also detected in a separate gene analysis. Accordingly, in an
embodiment, the method comprises detection of DNA copy number of a gene
in Tables 5-11 that falls within a MCR listed in Table 1, 2, 3, 4 and/or
10.

[0159] As a number of genome gains and losses are associated with tumour
responsiveness and/or better survival when subjects are treated with
chemotherapy, the disclosure provides methods for selecting a treatment
for subjects with lung cancer.

[0160] Accordingly, in another aspect, the disclosure provides a method of
selecting a treatment regimen for a subject with lung cancer, the method
comprising: (a) determining a genomic profile comprising detecting a
genomic alteration in one or more genes selected from Table 5 and/or 7 in
a biological sample from the subject; (b) selecting a treatment for the
subject optionally by comparing the genomic profile with one or more
controls, wherein the treatment selected comprises chemotherapy when the
genomic profile comprises a gain of all or part of one or more of the
following genes: MFSD7, D4S234E, ACOX3, SRD5A1, AQP2, ACCN2, SLC11A2,
SCN8A, KRT81, KRT1, ESPL1, NPFF, ATP5G2, HOXC11, NEUROD4, ZBTB39,
KIAA0286, INHBE, MARS, B4GALNT1, TSFM, DNMT3B; and/or a loss of all or
part of one or more of the following genes: RHOC, ATP2C2, ZDHHC7, COC4I1,
and/or FOXF1 relative to the control.

[0161] In another embodiment, the gain associated with improved survival
with chemotherapy or improved tumor responsiveness is a gain of all or
part of one or more of the following genes: BAALC, ANGPT1, MYC, WISP1,
KRT81, KRT1, NEUROD4, and/or PA2G4 (e.g. Table 9 genes associated with
improved response to chemotherapy). In a further embodiment, the gain
associated with improved survival with chemotherapy or improved tumor
responsiveness is a gain of all or part of one or more of the following
genes: GUCA2A, PPIH, LEPRE1, CR623026, C1orf50, DQ515898, DQ515897, MYC
FGF3, KRT81, KRT1, FAM112B, B4GALNT1, CENTG1, BCL11B (e.g. Table 11 genes
associated with improved response to chemotherapy).

[0162] In an embodiment, the gain comprises a gain in all or part of one
or more of FGF3, FAM112B, TSFM, NUP107 and/or MDM2.

[0163] In an embodiment, the subject has been treated by surgical
resection.

[0164] Two genes were identified as trending to worse survival with
administration of chemotherapy.

[0165] Accordingly, in an embodiment the method for selecting a treatment
comprises: (a) determining a genomic profile comprising detecting a
genomic alteration in one or more genes selected from AK024870 and CPSF6;
wherein the treatment selected comprises non-chemotherapy and/or a
non-platinum analog-, vinca alkaloid or combination thereof chemotherapy
treatment when the genomic profile comprises a gain of all or part of one
or more of AK024870 and CPSF6.

[0166] The disclosure also provides a method of prognosis of likelihood of
improved survival in a lung cancer subject who was and/or is receiving a
chemotherapeutic treatment, comprising determining the presence or
absence of a gain or loss of a MCR associated with improvement with
chemotherapy, predicting the likelihood of improved survival according to
the presence or absence of the MCR or gene gain or loss compared to a
control, wherein detecting a MCR or gene associated with improvement with
chemotherapy predicts likelihood of improved survival compared to a
control having the same gain or loss who has not received or is not
receiving chemotherapy.

[0167] In an embodiment, the presence of a gain or loss associated with
improvement with chemotherapy is indicative of a favourable
predisposition of the subject to respond to platinum analogs, vinca
alkyloids and/or a combination thereof.

[0168] Another aspect provides a method of treating lung cancer comprising
determining the presence or absence of a gain or loss of a MCR or gene
associated with improvement with chemotherapy in a subject with lung
cancer and administering chemotherapy to a subject with at least one gain
or loss associated with improvement with chemotherapy.

[0169] In an embodiment the chemotherapy administered is a platinum
analog, a vinca alkyloid or a combination thereof. In a further
embodiment, the platinum analog is selected from the group consisting of
cisplatin, paraplatin, carboplatin, oxaliplatin and satraplatin in either
IV or oral form. In another embodiment the vinca alkyloid is selected
from the group vinorelbine, vincristine, vinblastine, vindesine and
vinflunine in either IV or oral form.

[0170] The methods described herein are useful for different lung cancers.
In an embodiment, the lung cancer is non-small cell lung cancer (NSCLC),
early stage NSCLC, squamous cell carcinoma, adenocarcinoma, or large cell
carcinoma.

[0171] The biological sample can be any sample that comprises a
polynucleotide or biomarker expression product to be assayed. In an
embodiment, the biological sample is selected from the group consisting
of lung tissue, lung cells, lung biopsy and sputum, including formalin
fixed, paraffin embedded and fresh frozen specimens.

[0172] The methods described herein compare a subject profile, genomic or
expression with a control. The control with respect to genomic
alterations is for example the copy number of gene or region in a subject
in a different class e.g. good prognosis when treated with chemotherapy
versus poor prognosis when not treated with chemotherapy, or
alternatively can be an internal control, e.g. the copy number at a
region with no gain or loss, for example centromere copy number. For
example, For the FISH method, the centromere copy number can be used. For
the qPCR method, centromere cannot be used, and instead a "control" gene
would be used, a gene on the same or different chromosome that is
infrequently gained or lost. For array-CGH, a reference genomic DNA
sample from a "normal" individual without cancer would be used. A person
skilled in the art would be able to select an appropriate control.
Accordingly, in an embodiment the control is the centromere copy number.
Typically, the copy number of a gene or region is 2, one copy per allele.
Accordingly, in another embodiment the control is such that a copy number
greater than 2 is a gain, and a copy number less than 2 is a loss. Myc
and CCDN1 have for example, previously been shown to be amplified in lung
cancer, however it is not believed that they have been identified in
association with improved response to chemotherapy.

[0173] In an embodiment, for example pertaining to prognosis without
chemotherapy, the gene detected is not EGFR, MET, MYC, CCND1, KRAS,
and/or TITF1.

III. Compositions and Kits

[0174] The disclosure also provides compositions and kits which are useful
for example in the methods described herein.

[0175] An aspect provides a composition comprising a detection agent for
detecting the presence or absence of a MCR or gene gain or loss
associated with prognosis. In an embodiment the detection reagent is a
hybridization probe, for example a chromosomal probe or a gene expression
probe. In an embodiment, the probe comprises at least 8, 10, 15, 20, 25,
50, 75, 100, 150, 200, 250, 400, or 500 contiguous nucleotides
complementary to a gene listed in Table 5, 6, 7, 8, 9 and/or 11, or a
genomic region alteration such as a MCR and/or region flanking a MCR
described herein, for example in Tables 1 to 11, or for example in Table
1, 2, 3, 4 and/or 11. The probe can further be 90, 95, 96, 97, 98, 99,
99.5, 99.9% identical to the at least 8, 10, 15, 20, 25, 50, 75, 100,
150, 200, 250, 400, or 500 contiguous nucleotides of a gene listed in
Table 5, 6, 7, 8, 9 and/or 11, and/or a MCR and/or region flanking a MCR
described herein, for example in Table 1, 2, 3, 4 and/or 10. Depending on
the probe type (e.g. oligonucleotide or BAC clone), the nucleotide length
of the probe can vary, and in the case of a BAC clone can include
sequence in addition to the gene or MCR associated. In an embodiment, the
probe is a BAC clone. In an embodiment, the BAC clone is at least 50 000,
100 000, 150 000 or 200 000 nucleotides. In an embodiment the BAC clone
is about 150 000-200 000 nucleotides. BAC clones can be used for example
as probes in FISH and some array CGH platforms. In an embodiment, the
probe is complementary to a MCR described herein. In a further embodiment
the probe comprises a BAC clone that overlaps the MCR or gene gained or
lost. In an embodiment the probe comprises the nucleotide sequence of a
BAC clone of an Affymetrix U133A chip comprising a MCR or gene gain or
loss described herein as associated with prognosis. A person skilled in
the art on the basis on the teachings herein, such as the teachings in
the Examples, would be able to identify the probes that correspond to the
particular MCRs and genes.

[0176] In another embodiment, the composition comprises a primer or a
primer pair for amplifying a biomarker expression polynucleotide, or a
genomic region described herein. The primer is in an embodiment, 15-20,
21-30, 31-40, 41-50 or more than 50 nucleotides in length.

[0177] In an embodiment the composition further comprises a carrier.

[0178] In another aspect, the disclosure provides a kit for determining
lung cancer prognosis in a subject comprising for example a detection
agent or composition described herein. In an embodiment, the kit
comprises a chromosomal probe wherein the probe hybridizes all or part of
a MCR listed in Tables 1 to 11, for example in Table 1, 2, 3, 4 and/or 10
and/or all or part of a gene listed in Tables 5, 6, 7, 8, 9 and/or 11.

[0179] In another aspect, the disclosure provides a kit for determining
lung cancer prognosis in a subject, the kit comprising one or more gene
expression probes, wherein the set comprises a probe specific for a gene
expression product of a gene listed in Tables 5, 6, 7, 8, 9 and/or 11.

[0180] In an embodiment, the probes are labeled, for example, the probes
are fluorescently labeled. In other embodiment, the kit comprises
labeling reagents for example for labeling subject sample, e.g. subject
DNA.

[0181] In another embodiment, the probes are comprised in an array on a
solid support.

[0182] In an embodiment, the kit comprises reagents for FISH analysis of a
MCR or gene gain or loss described herein, and a control region such as a
centromere or gene on the same or different chromosome. For example, the
kit comprises a probe for a MCR or gene gain or loss described herein,
and a reference probe to the centromere or a gene on the same or
different chromosome, and labeling reagents for labeling the probe.

[0183] In another embodiment, the kit comprises reagents for CGH analysis
of a MCR or gene gain or loss described herein, for example, the kit
comprises an array with one or more probes for one or more MCRs or genes
gained or lost described herein and labeling reagents for labeling the
subject sample DNA.

[0184] In a further embodiment the kit comprises reagents for PCR such as
quantitative or multiplex PCR. For example the kit comprises a primer set
for amplifying all or part of a MCR or gene, or multiple MCRs or genes,
described herein associated with prognosis, as well as one or more primer
sets for identifying one or more control genes on the same or different
chromosomes.

[0185] In yet a further embodiment, the kit comprises a primer set and
probe for detecting an amplification product.

[0186] In a further embodiment, the kit comprises a positive and/or a
negative control. The control in an embodiment comprises normal reference
DNA for CGH or FISH based kits. A positive control comprises a tumour
that is known to have a gain or loss at the particular target being
assayed.

[0187] In yet a further embodiment, the kit further comprising
instructions that indicate prognosis is determined to be poor in the
absence of chemotherapy when a hybridization pattern of the chromosomal
probe or set of chromosomal probes indicates a gain in a MCR in for
example, chromosome 11 or 12 listed in Table 1, 2 and/or 10, or a gain in
a MCR comprising all or part of a gene listed in Table 5, 9 and/or 11
and/or a loss of the a MCR comprising all or part of a gene listed in
Table 7, relative to control; good when a hybridization pattern of one or
more chromosomal probes indicates a gain in a MCR comprising all or part
of a gene listed in Table 6 and/or a loss of the MCR comprising all or
part of a gene listed in Table 8. In another embodiment, the kit
comprises instructions that indicate prognosis is determined to good when
treated with chemotherapy when a hybridization pattern of the chromosomal
probe or set of chromosomal probes indicates a gain in a MCR comprising
for example, all or part of MFSD7, D4S234E, ACOX3, SRD5A1, AQP2, ACCN2,
SLC11A2, SCN8A, KRT81, KRT1, ESPL1, NPFF, ATP5G2, HOXC11, NEUROD4,
ZBTB39, KIAA0286, INHBE, MARS, B4GALNT1, TSFM, and/or DNMT3B, and/or a
loss in a MCR comprising all or part of RHOC, ATP2C2, ZDHHC7, COC4I1,
FOXF1, relative to a control. In another embodiment, the kit comprises
instructions that indicate prognosis is determined to be good when
treated with chemotherapy, when a hybridization pattern of a chromosomal
probe or set of chromosomal probes indicates a gain in a MCR comprising
for example a gain listed in Table 9 and/or 11 to be associated with poor
prognosis. In an embodiment, the instructions include direction for
comparing to a control. In an embodiment, the instructions include
direction and/or reagents for using a centromere copy number or other
chromosome as a control.

[0188] The following non-limiting examples are illustrative of the present
disclosure:

EXAMPLES

Example 1

Results

[0189] Array-CGH and RNA Microarray:

[0190] The chromosomal pattern of observed gains and losses by array-CGH
are in concordance with previous array-CGH and CGH studies in NSCLC,
including frequent gains at chromosome 1q, 3q, 5p, and 8q, and frequent
losses at 3p, 5q, 6q, 8p, 9p, 13q, and 17p. MCRs of DNA copy number
alteration encompass multiple genes known to be important in NSCLC,
including MYC, hTERT, and cyclin D1, as well as many potentially
important novel genes.

[0191] Upon integration of wide MCRs of gain with RNA expression
microarray data, there are 38 genes that, when gained in copy number,
were found to impart a significantly worse survival in the absence of
chemotherapy (p<0.05) (Table 5). These genes are found mostly on
chromosomes 12q and 5p. Of these 38 genes 22 were found to show a
significant improvement with chemotherapy by the interaction terms
analysis on the array-CGH dataset. Only one gene (RAB11FIP1) was found to
have a favourable effect on prognosis when gained (Table 6).

[0192] Within the wide MCRs of loss, 13 genes had a significant
deleterious effect on survival in the absence of chemotherapy,
predominantly found on chromosomes 1p, 13q, and 16q (Table 7). Of these,
6 genes were found to show a significant improvement with chemotherapy by
the interaction terms analysis. Eight genes, mostly on chromosome 6p,
showed an improved prognosis with loss of DNA material in one of the 3
analyses.

[0193] After removing known human copy number variations, 27 narrow MCRs
of gain and 19 narrow MCRs of loss across the genome were identified for
statistical analysis. After correcting for multiple testing, MCRs of gain
within a 6 Mb region of 12q were found to be significantly associated
with poor survival in the absence of chemotherapy (p<0.001,
q<0.05). When this region was examined for benefit of chemotherapy, a
significant improvement of survival was identified at one of these 12q
MCRs (interaction p<0.01), while the other 12q MCRs showed a trend
towards improved response to chemotherapy (Table 1). These associations
remained significant (p<0.05) in a multivariate model incorporating
known prognostic clinical factors (i.e., age, sex, stage, grade).
Approximately 25% of samples showed gains at these MCRs on 12q, which
were more common in squamous cell carcinomas (40%) than adenocarcinomas
(20%), and tended to be seen in older patients.

[0194] Other potential predictive associations arising from this analysis
that were not significant after multiple testing corrections included an
improved survival with chemotherapy for patients with gains at MCRs on 8q
(interaction p=0.02) and 11q (interaction p=0.08). The 11q gain showed
significant predictive ability in the multivariate model (interaction
p=0.02), whereas the MCR on 8q lost its predictive ability in the
multivariate model in this analysis.

[0195] One hundred and twenty-three focal high-amplitude MCRs were
identified from the 113 NSCLC samples interrogated by array-CGH. These
amplicons were found on all 22 chromosomes examined, and included
well-known amplified genes in NSCLC including EGFR, MET, MYC, CCND1,
KRAS, and TITF1. Twenty-six of these high-amplitude MCRs were found to be
well known copy number variations (CNVs) contained within the Database of
Genomic Variants (DGV). Eleven of these MCRs were selected for further
validation studies based on significant survival associations (Table 10).

Quantitative Polymerase Chain Reaction (qPCR):

[0196] There were 40 genes on chromosomes 5, 8, and 12 from the wide MCRs
analysis, that were tested by qPCR on the same samples. Of these, 6 genes
showed a significant (p<0.05) poor survival in the observation arm
associated with DNA copy number gains as detected by qPCR (Table 9). Five
of the genes showed a significant (p<0.05) improved outcome with
chemotherapy by interaction terms analysis (Table 9). These survival
associations were in agreement with the array-CGH analysis. However, the
remainder of the genes tested did not show the same survival association
by qPCR as by array-CGH, on DNA from the same samples.

[0197] Upon examination of the minority of genes that were validated by
qPCR, it was noted that these genes tended to fall in regions that showed
high-level amplifications. As a result of this finding, an array-CGH
analysis designed to focus on high-level amplifications was performed,
resulting in the list of high-amplitude MCRs listed in Table 10.

[0198] From the 11 prognostic/predictive high-amplitude MCRs, 38 genes
have been tested by qPCR on the same samples. Of these, 16 have shown
significant (p<0.05) survival associations (prognostic in the absence
of chemotherapy, and/or predictive of improved response to chemotherapy)
in agreement with the array-CGH analysis. An additional 9 of these genes
show a trend to significant survival associations (p<0.2). Many of the
genes with significant survival associations were found within the four
12q amplicons, showing a poor prognosis in the observation arm, and an
improved response to chemotherapy.

Discussion

[0199] High-resolution array-CGH analyses on a subset of the BR 10
patients have identified regions of recurrent copy number gain that may
be predictive of benefit from adjuvant chemotherapy. This information
would be very useful for selecting those lung cancer patients who should
receive current adjuvant chemotherapy, those who do not require
chemotherapy, and those patients who will require more experimental
treatments in hopes of curing their disease. Further experiments are
underway to validate these results in additional samples from the same
study, as well as to identify critical genes in these areas. (Supported
by grants from the Canadian Cancer Society, Ontario Institute of Cancer
Research and Genome Canada)

Materials and Methods

Study Materials:

[0200] All NSCLC samples used in this study were excised from patients who
were enrolled in a prospective, randomized controlled trial (JBR10) which
studied the efficacy of adjuvant venorelbine plus cisplatin to improve
survival in early stage (stage IB or II) NSCLC patients who had been
treated by complete surgical resection (Winton et al., 2006). Half of the
patients were randomly assigned to receive adjuvant chemotherapy, and
half were assigned to no adjuvant chemotherapy. The samples examined were
excised prior to any adjuvant therapy being administered. The study
concluded that adjuvant chemotherapy prolongs disease free survival and
overall survival in patients with completely resected early-stage NSCLC.

[0201] For array-comparative genomic hybridization (CGH) analysis, DNA was
extracted from 134 formalin-fixed, paraffin-embedded (FFPE) and 16 fresh
frozen NSCLC specimens, from 142 patients. The FFPE samples were cored
from tissue blocks in areas of >60% tumour cells, as marked by a
pathologist on hematoxylin and eosin (H&E) slides.

[0202] For gene expression microarray experiments, 176 fresh frozen tumour
samples and 10 fresh frozen corresponding normal lung samples were used.
133 of these tumour samples were from patients in the JBR10 cohort, 81 of
which also had array-CGH data analyzed in this study. 38 of the tumour
samples were from a non-JBR10 cohort.

Array-CGH hybridization:

[0203] Array comparative genomic hybridization (CGH) was performed using a
custom whole genome tiling path bacterial artificial chromosome array
with 26,363 overlapping clones, each spotted in duplicate (BC Cancer
Research Centre, Vancouver, BC) (Watson S K et al., 2007). This platform
enables us to measure alterations in DNA copy number at high resolution
across the entire genome in each tumour sample, with a minimal amount (as
little as 50 ng) of DNA.

[0206] Data was log 2 transformed, and replicate clones having standard
deviations >0.075 or signal-to-noise ratios in each dye channel of
<3 were filtered out. A multi-step normalization was then carried out
to control for biases caused by the array (ex. spatial biases or
differences in background signal), the dyes used for labeling, or the DNA
sample quality (Khojasteh et al. 2005, Chi et al. 2007). The amount of
"copycat" correction required for each sample was plotted in a histogram
of all samples; those that required too much correction and did not lie
within a normal distribution were deemed to be poor quality DNA, and were
eliminated from analysis. By this criteria, 35 samples were eliminated,
leaving 115 samples from 113 patients (56 received adjuvant chemotherapy,
57 had no adjuvant chemotherapy) for further analysis. Log 2 ratios were
plotted and data was visualized using SeeGH software (Chi et al. 2004).

Array-CGH Data Analysis:

[0207] In order to define genomic regions that were frequently gained in
terms of DNA copy number in NSCLC, three algorithms were employed in
parallel analyses to define the segmental DNA gains and losses in each
tumour genome for the 113 patient samples: circular binary segmentation
(DNAcopy) (Venkatraman & Olshen, 2007), a hidden markov model (HMMeR)
(Shah et al., 2006), and aCGH Smooth (Jong et al. 2004). For DNAcopy
analysis, a log 2 threshold of 0.05 for gains and -0.05 for losses was
used to define whether a segment was gained/lost or not. For each
algorithm, minimal common regions (MCRs) of DNA gain and loss were then
identified for the entire tumor panel with STAC software (Diskin et al.
2006) (using 100 permutations at a resolution of 100,000 bp, and a
p-value cut-off of 0.05 by either footprint or frequency calculation by
the software). These regions are referred to herein and accompanying
tables as "wide MCRs of gain" and "wide MCRs of loss."

[0208] To attempt to focus further the genomic regions of DNA copy number
gain in NSCLC, circular binary segmentation (DNAcopy) (Venkatraman &
Olshen, 2007) was used to define the segmental DNA gains and losses in
each tumour genome for the 113 patient samples. A log 2 threshold of 0.05
for gains and -0.05 for losses was used to define whether a segment was
gained/lost or not. Minimal common regions (MCRs) of DNA gain and loss
were then identified for the entire tumor panel with STAC software
(Diskin et al. 2006) (using 100 permutations at a resolution of 100,000
bp) with a p-value cut-off of 0.05 by frequency calculation. MCRs
corresponding to known copy number variations as described by Wong et al.
2007 were eliminated. As well, MCRs whose frequency of alteration amongst
the samples multiplied by their average log 2 of altered samples was less
than 0.02 were removed from further analysis. These MCRs are referred to
as "narrow MCRs of gain" and "narrow MCRs of loss" herein.

[0209] In order to focus the array-CGH analysis on high-level
amplification events in NSCLC, circular binary segmentation (DNAcopy)
(Venkatraman & Olshen, 2007 was used to define the segmental DNA gains
and losses in each tumour genome for the 113 patient samples. A log 2
threshold of 0.05 was used to define whether a segment was gained or not.
High-amplitude regions of gain (referred to as "high-amplitude MCRs"
herein) were defined as genomic regions where the average log 2 value, as
assigned by DNAcopy analysis, in the gained samples, was greater than
0.15.

[0210] Prognostic and predictive genes by RNA expression levels within
MCRs of gain were determined by integrating data from gene expression
microarray experiments. Gene expression for 133 NSCLC samples was
assessed using an Affymetrix U133A microarray chip. The data was
normalized using RMAexpress software followed by distance-weighted
discrimination (DWD) to minimize "batch" differences among samples, and
then log 2 transformed.

Statistical Analysis:

[0211] In order to identify prognostic genes, the MCRs of gain and loss as
defined above (p-value 0.05 by frequency or footprint calculation) were
cross-referenced with the locations of genes on the Affymetrix U133A chip
(˜22,000 probesets in total) that were found to have prognostic
value by univariate Cox proportional hazards analysis on the observation
arm only. Out of 1584 probesets that had a significant prognostic effect
(p<0.05) by gene expression, 398 probesets (364 genes) fell within
MCRs of gain, and 426 probesets (391 genes) fell within MCRs of loss.
These genes were selected for further analysis.

[0212] To evaluate the prognostic significance of genomic gain or loss at
each of the genes in the absence of adjuvant therapy, a univariate Cox
proportional hazards model using disease-specific survival (DSS) was
applied to determine any statistically significant (p<0.05) prognostic
effect for the patients who did not receive chemotherapy (57 patients).
Hazard ratios were compared to ensure agreement between the gene
expression and array-CGH data in terms of the effect on patient survival,
and 4 lists of genes were arrived at: genes imparting a worse prognosis
when gained (39 genes), genes imparting a better prognosis when gained (1
gene), genes imparting a worse prognosis when lost (13 genes), and genes
imparting a better prognosis when lost (8 genes).

[0213] In addition, a univariate Cox proportional hazards model was
employed on the entire cohort (observation and chemotherapy arm, 113
patients in total) with the use of interaction terms to identify effects
of chemotherapy on the survival associated with gain or loss at each
gene.

[0215] To examine any clinicopathological associations between genomic
gains and losses at each MCR, a Fisher's exact test was employed, using
sex, nodal status, and histologic cell type as variables.

Quantitative Polymerase Chain Reaction (qPCR):

[0216] Quantitative PCR was performed using the SYBR Green method and the
Roche Lightcycler 480 instrument. Five ng of genomic DNA were used per
well in triplicate in 384 well plates. Primers were designed and tested
for specificity using the online Primer Blast software (NCBI). Primers
were designed to target one exon region of each gene, with a bias towards
3' exon location. As a reference, primers were designed for 3 genes on
different chromosomes that are infrequently altered numerically in NSCLC,
as guided by our array-CGH results. Dissociation curves (melting curves)
for each primer pair were determined to test for contamination,
mispriming, and primer-dimer artifact; only primers producing a single
peak in the dissociation curve were used in the assays.

[0217] Standard curves were derived using pooled DNA from 20
formalin-fixed paraffin-embedded lung tissue from resection specimens,
taken from blocks uninvolved by tumour. In addition, 23 normal FFPE lung
samples were run along with the tumour samples in each reaction.

[0218] Initial processing of data was carried out using the Roche
Lightcycler 480 software, which calculates using the 2nd derivative
max point to determine crossing-point (CP) values for each well. CP
values were mapped to the standard curve for each gene to obtain DNA
concentration values for each well. The gene copy number was normalized
against the copy number of the reference genes. A normal range of gene
copy number for each gene was established with the 23 samples of
non-neoplastic lung DNA, and samples with copy number 2sd above the mean
were identified as gained in copy number. Samples with copy numbers, as
calculated by advanced relative quantification, of greater than 4, were
identified as having an amplification (in addition to a gain) at that
gene, by qPCR analysis.

Example 2

Selection of Genes for Quantitative PCR Validation

[0219] Genes within wide MCRs of gain on chromosomes 5, 8, and 12 that
showed concordant survival effect by transcript level and DNA copy number
were chosen for the first round of quantitative PCR validation.

[0220] For the second round of quantitative PCR validation, 5 genes within
each prognostic/predictive high-amplitude MCR were selected by ranking
them using the following criteria: RNA expression data showing the same
survival effect for the RNA transcript quantity as for the DNA copy
number, gene ontology relating to oncogenicity, average log 2 ("raw" log
2 values as well as log 2 values assigned by DNAcopy) among gained
samples, STAC analysis frequency p-value<0.05, overexpression of RNA
transcripts in NSCLC, location within an amplicon reported previously in
the literature, p-values of prognostic and predictive survival
associations for DNA copy number at that location (both univariate and
multivariate), and p-values for prognostic and predictive survival
associations of RNA transcript levels (univariate).

Example 3

[0221] The array-CGH dataset described in Example 1 is unique and powerful
in that it uses tumour samples from a randomized controlled trial of the
effectiveness of chemotherapy in early-stage NSCLC, providing an
unprecedented opportunity to study genomic aberrations at high-resolution
and correlating them with patient outcome in the presence or absence of
chemotherapy. The sample size (113) is more than double the majority of
previous array-CGH studies, allowing for a greater power in determining
prognostic and predictive effects of gains and losses. Furthermore, the
resolution of our platform is superior to most previous array-CGH studies
in NSCLC, allowing us to more precisely define the breakpoints of
amplifications and deletions. An additional 180 samples from the same
trial will be processed to further validate the survival associations
found in the array-CGH study described herein.

Example 4

Optimization of the Prognostic and Predictive Gene Copy Number Model

[0222] The gains and losses outlined herein could be tested for
associations amongst one another using methods of multivariate statistics
including but not limited to, cluster analysis, principal component
analysis, and logistic regression. In this way, copy number alterations
that tend to occur together could be identified, and key alterations that
could serve as surrogate biomarkers for the co-occurring events could be
identified. These key copy number alterations could be incorporated into
a weighted model or that could be used to identify one or more "copy
number signatures" that could molecularly classify non-small cell lung
carcinomas. Such a signature would be useful for predicting prognosis and
response to chemotherapy.

Example 5

[0223] The sample of lung tumour is obtained during surgery or a minimally
invasive procedure. The tissue is processed in the lab to identify the
tumour content. A portion of the tumour is frozen, or fixed in formalin
and embedded in paraffin as per standard laboratory protocol. The DNA is
extracted from the tumour tissue, and subjected to a laboratory test to
examine for specific genomic alterations, such as array-CGH or multiplex
qPCR. Alternatively, sections are cut from a paraffin block containing
tumour, and processed for FISH analysis using probes hybridizing to one
or more of our targets, and the tumour nuclei are scored for gains and
losses. The presence as determined by these tests of a gain or loss in
copy number, compared to a control (internal or external, depending on
the test), indicates a poor prognosis for the patient if not treated with
chemotherapy, but a significantly improved prognosis if treated with
chemotherapy.

Example 6

[0224] How to identify probes used herein useful for detecting gains and
losses associated with prognosis.

[0225] An individual could take the known genomic location of the MCR and
then apply online resources to determine which BAC clones span the
recurring alteration (e.g. Human BAC
Resource--http://www.ncbi.nlm.nih.gov/genome/cyto/hbrc.shtml). SMRT array
mapping information--specific to individual BAC clones--is available
online (http://www.bccrc.ca/cg/ArrayCGH_Group.html,
http://bacpac.chori.org/order.php).

[0226] Individuals can take the known genomic location, open the mapping
file, and determine which BAC clones span the MCR region they are
interested in. Individuals could then order clone(s) for their own use
from an online resource (e.g. BACPAC Resources Center
http://bacpac.chori.org/order.php). Labeled probes from this DNA could
then be made and applied using a standard FISH protocol. Alternatively,
labeled probes for FISH from a given clone could also be ordered directly
from a variety of sources, including the BC Cancer Research Centre
(http://arraycgh.ca/services.php).

[0228] While the present disclosure has been described with reference to
what are presently considered to be the preferred examples, it is to be
understood that the disclosure is not limited to the disclosed examples.
To the contrary, the disclosure is intended to cover various
modifications and equivalent arrangements included within the spirit and
scope of the appended claims.

[0229] All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as if each
individual publication, patent or patent application was specifically and
individually indicated to be incorporated by reference in its entirety.